Cell viability compensation (CVC) beads for flow cytometry applications and methods of use thereof

Abstract

The present disclosure provides control compositions and methods of making and using control compositions. Aspects of a control composition 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 for binding to the DNA binding dye wherein the aminated polymer and the nucleic acid are covalently bound to the core. Aspects of the invention further include a method of synthesizing a control particle of a composition, the method of synthesis comprising: 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 of compensating for cell viability data obtained using, for example, flow cytometry using the control compositions, as well as kits for practicing the subject methods.

Description

Cross - Reference to Related Applications

[0001] This application claims the benefit of the filing date of U.S. Provisional Patent Application Serial No. 63 / 627,756, filed on January 31, 2024, the disclosure of which is incorporated herein by reference. Background Art

[0002] Optical detection is often used to characterize the components of biological samples. When a sample is irradiated, light can be scattered by the sample, transmitted through the sample, or emitted by the sample (e.g., by fluorescence). Changes in sample components, such as morphology, absorbance, and the presence of fluorescent labels, can cause changes in the light scattered, transmitted, or emitted by the sample. To quantify these changes, light is collected and directed onto the surface of a detector. One technique that uses optical detection to characterize sample components is flow cytometry.

[0003] In flow cytometry applications, viability staining has been used to distinguish between live and non - live mammalian cells based on fluorescence intensity. Viability staining works because there are differences between the cell membranes of necrotic (i.e., non - live) cells and healthy (i.e., live) cells. In necrotic cells, the damaged cell membrane allows dyes to enter the cell interior and interact with various intracellular components, while the intact cell membrane of healthy cells prevents most dyes from entering the cell.

[0004] Currently, viability staining mainly employs two general techniques: staining assays based on amine - reactive fluorescent dyes and staining assays based on DNA - intercalating dyes. For assays based on amine - reactive dyes, the reactive dyes can enter the membranes of non - live cells and react with a large number of free amines on internal proteins, thereby strongly fluorescently labeling the non - live cells of the sample (i.e., relative to live cells). Similarly, for assays based on DNA - intercalating dyes, the DNA - binding dyes can cross the cell membranes of non - live cells and enter and react with the DNA in the cell nucleus, thereby producing a relatively strong fluorescent label for non - live cells.

[0005] Like other sample characterization techniques using flow cytometry, viability staining assays require controls in order to set voltage and compensation parameters and obtain accurate and reproducible results. Unfortunately, most current viability staining protocols require the experimenter to consume a portion of the usually limited cell sample to create the necessary viability control. In addition, creating such controls is often time - consuming and unreliable, failing to achieve the brightness discrimination between live and dead cells required to obtain meaningful results. Summary of the Invention

[0006] Accordingly, there is a need for improved and useful control compositions for cell viability assays. Embodiments of the present invention provide such useful new control compositions and methods of use thereof to address the above limitations. To achieve this, embodiments of the present invention utilize recent advances in materials science and biochemistry, as well as novel particle coating techniques, to effectively synthesize novel, convenient, and effective control particles. The coating technique of the present disclosure utilizes a unique layer-by-layer method to allow the creation of highly versatile compensation particles capable of binding both amine-reactive dyes and DNA intercalating dyes. This versatility, combined with the high tunability of the disclosed particle synthesis method, enables the simplification of current laboratory workflows while also providing additional flexibility to users in developing future experimental designs. Accordingly, embodiments of the disclosed compositions and methods (e.g., as described in more detail below) can be used in a variety of applications in which it is desired to accurately and adaptively perform cell viability assays at low cost and high efficiency.

[0007] In one aspect, control particles for performing cell viability assays are provided. Aspects of the control particles include: a core; a polymer that includes amines configured to react with amine-reactive dyes (i.e., an aminated polymer); 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.

[0008] In certain embodiments, the core of the control particles is substantially free of autofluorescence. In some embodiments, the core has low non-specific binding. In some embodiments, the core includes a polymer, such as poly(methyl methacrylate) (PMMA) or a polyacrylamide hydrogel. In some embodiments, the core includes an inorganic material, such as silica (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 bonded to the surface of the core directly or via a linker, and the aminated polymer is covalently bonded to the nucleic acid. In some embodiments, the aminated polymer and the nucleic acid are layered on the core. In some embodiments, multiple layers of the aminated polymer and / or multiple layers of the nucleic acid of the coating layer are covalently associated with the core. In some embodiments, the particles include 5 or more layers of the aminated polymer and / or 5 or more layers of the nucleic acid. In some embodiments, the layers of the aminated polymer of the coating layer alternate with the layers of the nucleic acid of the coating layer. In some embodiments, the control particles are beads.

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

[0010] In certain embodiments, the size of one or more nucleic acids of the control particles ranges from 300 base pairs to 700 base pairs. In some embodiments, one or more nucleic acids of the control particles each comprise 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, one or more nucleic acids are configured to bind to a DNA-binding dye freely flowing in solution. In some embodiments, the maximum emission wavelength range of the DNA-binding dye is from 400 nm to 850 nm. In some embodiments, the maximum absorption (maximum excitation) range of the DNA-binding dye is 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-diamidino-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 comprise one or more polyethylene glycol (PEG) compounds configured to prevent reaction of amine-reactive dyes with amines of the negative control particles. In some embodiments, the negative control particles do not comprise an aminated polymer or nucleic acid; for example, the negative control particles consist of a core.

[0012] In certain embodiments, a control composition comprising multiple control particles is provided. In some embodiments, the control composition comprises multiple unlabeled positive control particles. In some embodiments, the unlabeled control composition further comprises multiple negative control particles. In some embodiments, the control composition comprises multiple labeled positive control particles. In some embodiments, the labeled control composition further comprises multiple negative control particles. In some embodiments, the control composition comprises a liquid solution, such as a buffer. In some embodiments, the control composition is lyophilized.

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

[0014] In certain embodiments, the surface of the core comprises functional groups, such as amide, maleimide or thiol groups. In some embodiments, the method further comprises surface-functionalizing the core with a functional group. For example, the obtained core may comprise silica, and the functionalization may include amination. In some embodiments, the method further comprises modifying the functional group of the core with a reagent, such as a reagent, to comprise a reactive functional group. For example, a thiolation reagent such as 2-iminothiolane may be used to modify an aminated silica core to comprise a thiol group. In some embodiments, the method further comprises making the core positively charged, for example, to increase the efficiency of binding to nucleic acids before associating the aminated polymer with the core. In some embodiments, the method further comprises making the core negatively charged, for example, to increase the efficiency of binding to the aminated polymer before associating the nucleic acid with the core.

[0015] In certain embodiments, the nucleic acid comprises deoxyribonucleic acid (DNA), such as double-stranded DNA (dsDNA). In some embodiments, the particle synthesis method further comprises modifying the DNA to include functional groups that react with the functional groups of the core and / or the functional groups of the aminated polymer. For example, the core and / or the aminated polymer can be modified to include thiol groups, and the DNA can be modified to include maleimide groups. In some embodiments, the nucleic acid is covalently associated with the core by reacting the functional groups of the DNA with the reactive functional groups of the core. In some embodiments, for example, when the aminated polymer has been covalently associated with the core, the nucleic acid is covalently associated with the core by reacting the functional groups of the DNA with the functional groups of the aminated polymer. In some embodiments, the 5' end and / or the 3' end of the DNA is functionalized. In some embodiments, the DNA is dsDNA, and both strands of the dsDNA are functionalized. In some embodiments, the dsDNA is covalently bonded to the core by reacting the functional groups of the first strand of the dsDNA with the reactive functional groups of the core, and the dsDNA is covalently bonded to the aminated polymer by reacting the functional groups of the second strand of the dsDNA with the functional groups of the aminated polymer. In some embodiments, both strands of the dsDNA are covalently bonded to the aminated polymer.

[0016] In certain embodiments, the particle synthesis method further comprises modifying the aminated polymer to include functional groups that react with the functional groups of the core and / or the functional groups of the nucleic acid. For example, the core and / or the nucleic acid can be modified to include maleimide groups, and the aminated polymer can be modified to include thiol groups. In some embodiments, the aminated polymer is covalently associated with the core by reacting the functional groups of the aminated polymer with the reactive functional groups of the core. In some embodiments, for example, when the nucleic acid has been covalently associated with the core, the aminated polymer is covalently associated with the core by reacting the functional groups of the aminated polymer with the functional groups 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 bonded to the core by reacting the first functional group of the aminated polymer with the reactive functional groups of the core, and the aminated polymer is covalently bonded to the nucleic acid by reacting the second functional group of the aminated polymer with the functional groups of the nucleic acid. In some embodiments, two or more functional groups of the aminated polymer are covalently bonded to the nucleic acid.

[0017] In certain embodiments, the aminated polymer and the nucleic acid are covalently associated with the core by alternating layers of the coated layers of the aminated polymer and the nucleic acid. For example, the nucleic acid can include dsDNA, and two or more nucleic acids can be modified to include maleimide groups at the 5' ends of each strand of the dsDNA, the aminated polymer can include a protein, and two or more aminated polymers can be modified to include two or more thiol groups, and the core can include a plurality of thiol groups. Then, the layering can 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, 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, 5 or more rounds of layering are performed such that the control particles include 5 or more nucleic acid layers and 5 or more aminated polymer layers. In some embodiments, each round of layering includes covalently associating a plurality of nucleic acids and a plurality of aminated polymers to the core.

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

[0019] In another aspect, a method of 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 is provided. Aspects of the method include: analyzing a labeled compensation control composition using a flow cytometer to obtain flow cytometry data; and calculating a 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 certain embodiments, preparing includes contacting an 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 compensate cell viability data. In some embodiments, compensation is performed by: contacting a cell sample with the same dye bound to labeled positive control particles; generating cell viability data by analyzing the cell sample using a particle analyzer (e.g., a flow cytometer); modifying the cell viability data using the determined compensation value.

[0021] In another aspect, a kit is provided that includes a compensation control composition as described herein. In some embodiments, the kit further includes an amine-reactive dye and / or a DNA-binding dye for labeling positive control particles of the compensation control composition. In some embodiments, the kit further includes instructions for determining a compensation value for cell viability data obtained from flow cytometry analysis using the compensation control composition. In some embodiments, the kit further includes instructions for performing flow cytometry analysis and compensating the obtained cell viability data using the determined compensation value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Descriptions of two types of viability staining mechanisms according to embodiments of the present invention are provided.

[0023] Figure 2 An illustration of cell viability compensation (CVC) beads according to embodiments of the present invention is depicted.

[0024] Figure 3 An exemplary method for synthesizing CVC beads according to embodiments of the present invention is shown.

[0025] Figure 4 Staining results of prior art compensation beads and CVC beads synthesized according to embodiments of the present invention for the BD Horizon™ FVS520 amine-reactive dye are provided.

[0026] Figure 5 Staining results of prior art compensation beads and CVC beads synthesized according to embodiments of the present invention for the BD Horizon™ FVS570 amine-reactive dye are provided.

[0027] Figure 6 Staining results of prior art compensation beads and CVC beads synthesized according to embodiments of the present invention for the BD Horizon™ FVS700 amine-reactive dye are provided.

[0028] Figure 7Provided are the staining results of compensation beads of the prior art and CVC beads synthesized according to an embodiment of the present invention for two DNA intercalating dyes - BD Pharmingen™ 7-AAD and BD Pharmingen™ DAPI.

[0029] Figure 8 Provided are the results of a stability study conducted on CVC beads synthesized according to an embodiment of the present invention.

[0030] Figures 9A to 9B Provided is a schematic diagram of an exemplary particle sorting system for CVC beads of the present invention according to certain embodiments.

[0031] Figure 10 Depicted is a functional block diagram of an exemplary particle analyzer control system for CVC beads of the present invention according to certain embodiments.

[0032] Figures 11A to 11B Shown is an exemplary particle analysis system for CVC beads of the present invention according to certain embodiments. Figure 11A Depicted is a flow cytometer of the particle analysis system. Figure 11B Depicted is a functional block diagram of the particle analysis system.

[0033] Figures 12A to 12B Shown are exemplary particle sorters and data processing techniques for performing viability staining assays using radiofrequency tagging emission for fluorescence imaging according to certain embodiments. Figure 12A Provided is a schematic diagram of an image-enabled particle sorter. Figure 12B Provided is a flowchart depicting data processing techniques.

[0034] Figure 13 Depicted is a block diagram of a computing system for CVC beads of the present invention according to certain embodiments. Detailed Description

[0035] The present disclosure provides control compositions and methods of making and using the control compositions. Aspects of the control compositions include control particles, characterized by: a core; a polymer comprising amines configured to react with amine-reactive dyes; and nucleic acids configured to bind to DNA-binding dyes, wherein the aminated polymer and the nucleic acids are covalently associated with the core. Aspects of the present invention further include a method of synthesizing control particles of the composition, the synthesis method comprising: obtaining a core; and covalently associating a polymer comprising amines and nucleic acids with the core to produce control particles. Also provided are methods of compensating cell viability data obtained using a particle analyzer (e.g., a flow cytometer) using the control compositions, and kits for practicing the subject methods.

[0036] Before describing the present invention in more detail, it is to be understood that the invention is not limited to the particular embodiments described, and thus may of course vary. It should also 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 invention will be limited only by the appended claims.

[0037] Where a numerical range is provided, it is to be understood that each intermediate value between the upper and lower limits of that range (down to one-tenth of the unit of the lower limit unless the context clearly dictates otherwise) as well as any other stated value or intermediate value in the stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specific exclusions stated in the stated range. Ranges excluding one or both of the included limits of the stated range are also included in the invention when the stated range includes one or both of the limits.

[0038] Certain ranges of values presented herein are preceded by the term "about". The term "about" is used herein to provide literal support for the exact number that follows as well as for a number that is close to or approximate to the number that follows. When determining whether a number is close to or approximate to a specifically recited number, an unrecited number that is close to or approximate to the specifically recited number may be a number that provides a substantial equivalent in the context presented.

[0039] Unless defined otherwise, 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. Representative illustrative methods and materials are now described, although any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention.

[0040] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe the methods and / or materials associated with the publication citation. 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 a prior invention. Further, the provided publication dates may be different from the actual publication dates, which may require independent verification.

[0041] It should be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as antecedent basis for the recitation of claim elements using exclusive terms such as "solely", "only", etc., or using "negative" limitations.

[0042] As will be apparent to those skilled in the art upon reading this disclosure, each of the various embodiments described and illustrated herein has discrete components and features that can be readily separated from or combined with the features of any of several other embodiments without departing from the scope or spirit of the invention. Any of the recited methods can be performed in the order of the recited events or in any other order that is logically possible.

[0043] For purposes of grammatical fluency and functional explanation, the apparatus and methods have been or will be described, but it should be understood expressly that the claims should not be construed as necessarily limited in any way by an interpretation of the limitations of "apparatus" or "step" unless expressly recited under 35 U.S.C. § 112, but rather should be accorded the meaning of the definition provided in the claims and the full scope of equivalents under the doctrine of equivalents, and the cases where the claims are expressly recited under 35 U.S.C. § 112 should be accorded all statutory equivalents under 35 U.S.C. § 112.

[0044] As described above, control compositions are provided. In further describing the various embodiments of the invention, the subject compositions are first described in more detail, including, for example, control particles of the compositions. Next, methods for synthesizing control particles of the compositions are described. In addition, methods for compensating cell viability data using the control compositions are provided, as well as kits for practicing the subject methods.

[0045] Control compositions As described above, control compositions are provided, such as flow cytometry compensation control compositions. Control compositions can be used to verify the function or performance of particle analysis systems (e.g., flow cytometry systems), protocols, and reagents for use in flow cytometry assays. In certain embodiments, control compositions can be used to interpret flow cytometry data.

[0046] In some embodiments, the control compositions are used for 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 a flow cytometer except for the channel / detector specifically used to measure the fluorescent particle. In some cases, the subject control compositions (e.g., cell viability compensation [CVC] beads) can be used for fluorescence compensation of flow cytometry cell viability assays in order to, for example, distinguish live cell populations from dead cell populations in flow cytometry data. In some embodiments, flow cytometry cell viability assays use amine-reactive dyes to distinguish live cells from dead cells. In certain embodiments, flow cytometry cell viability assays use DNA-binding dyes to distinguish live cells from dead cells.

[0047] 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) for a given assay. In some embodiments, the positive control particles are configured to bind to fluorophores for both an amine-reactive fluorogenic 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 fluorogenic 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 amine-reactive fluorogenic dyes and DNA intercalating dyes. 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 tagged nucleic acid probes in addition to amine-reactive fluorogenic dyes and DNA intercalating dyes.

[0048] As described above, embodiments of the control composition include positive control particles. The positive control particles can comprise or be characterized by: a core; a polymer comprising an amine (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, wherein the aminated polymer and the nucleic acid are covalently associated with the core. In some embodiments, the core of the positive control particles comprises a material having low autofluorescence and / or low non-specific binding. In some embodiments, e.g., when considered as a whole, the core of the positive control particles is substantially non-autofluorescent and / or exhibits low non-specific binding. In some embodiments, the aminated polymer and the 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 can be relatively uniformly dispersed on the surface of the core to form a coating layer on the core. In some cases, the nucleic acid molecules and the aminated polymer molecules form separate, alternating coating layers on the surface of the core. In these cases, the positive control particles can comprise multiple layers of each of the aminated polymer and the nucleic acid molecules, e.g., 2 or more layers, or 3 or more layers, or 4 or more layers, or 5 or more layers, or 10 or more layers, etc., of each. In some cases, the positive control particles comprise a number of layers sufficient to bind a certain amount of the amine-reactive dye and / or the DNA-binding dye (e.g., for a given cell viability assay) such that the positive control particles are at least as bright as the cells stained with the same dyes. In these cases, the positive control particles can comprise a number of layers sufficient to bind the same or a greater amount of the amine-reactive dye and / or the DNA-binding dye as non-viable cells (e.g., non-viable mammalian tissue cells grown under specific conditions) in a particular cell viability assay. In some embodiments, the positive control particles can be configured to have substantially the same autofluorescence as the cells in 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 positive control particles are beads.

[0049] In certain embodiments, the control compositions of the present disclosure can include negative control particles. In some cases, the negative control particles include: a core. In some cases, the core of the negative control particles can be substantially the same as the core of the positive control particles. For example, the core of the negative control particles can include the same material, the same size and / or shape, and have the same properties (e.g., autofluorescence properties) as the core of the positive control particles. In some embodiments, the core of the negative control particles comprises a material having low autofluorescence and / or low non-specific binding. In some embodiments, for example, when considered as a whole, the core of the negative control particles substantially does not autofluoresce and / or exhibits low non-specific binding. In certain embodiments, the negative control particles do not include an aminated polymer or nucleic acid (i.e., do not include any aminated polymer molecules or any nucleic acid molecules). In these cases, the core of the negative control particles can be conjugated to 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 (e.g., PEG, poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), etc.) layer, can be deposited on the surface of the core of the negative control particles. In some embodiments, the negative control particles are configured to have substantially the same autofluorescence 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 can be mammalian cells. In some cases, the negative control particles are beads.

[0050] A control composition (e.g., a CVC composition) can include a plurality of positive control particles. In some embodiments, the control composition can include a plurality of negative control particles. In some cases, the control composition can include a plurality of positive and negative control particles. In certain embodiments, the control composition can include positive control particles labeled (e.g., bound / associated) with an amine-reactive dye and / or a DNA-binding dye. In some embodiments, the control composition can be suspended in a solution (e.g., a liquid buffer). In other cases, the control composition can substantially not contain a liquid (e.g., the control composition can be dehydrated or lyophilized).

[0051] In certain embodiments, the control compositions of the present disclosure are storage-stable control compositions, where the control compositions are substantially stable over an extended period of time. "Stable" or "storage-stable" or "substantially stable" means that the control compositions containing control particles retain their properties (e.g., autofluorescence, binding properties, etc.) and do not significantly reduce / alter their reactivity over an extended period of time. For example, stable positive control particles can retain their ability to react with amine-reactive dyes and / or bind to DNA-binding dyes, while negative control particles can retain their inertness 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 result in a significant change in dye-binding activity (the dye-binding activity of the negative or positive control particles of the composition) due to degradation of the control composition over an extended period of time. Such stability can be reflected by no change or a negligible change in the fluorescence of the positive and / or negative control particles of the control composition upon staining with a specific fluorescent particle (e.g., an amine-reactive dye or a DNA-binding dye) over an extended period of time, such as a change in fluorescence 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 change in fluorescence activity. In some cases, the storage-stable control composition has a change in fluorescence activity (e.g., dye-binding activity) of 5% or less over an extended period of time.

[0052] In some cases, a storage-stable control composition substantially retains its binding activity for fluorescent particles (e.g., amine-reactive dyes and / or DNA-binding dyes) 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, a storage-stable control composition can retain 90% or more of its binding activity over an extended period of time. In some cases, a storage-stable composition retains 95% or more of its binding activity over an extended period of time. The extended period of time is, for example, 1 week or longer, or 2 weeks or longer, or 3 weeks or longer, or 1 month or longer, or 2 months or longer, or 3 months or longer, or 4 months or longer, or 6 months or longer, or 9 months or longer, or 1 year or longer, or 1.5 years (e.g., 18 months) or longer, or 2 years or longer, or 2.5 years (e.g., 30 months) or longer, or 3 years or longer, or 3.5 years (e.g., 42 months) or longer, or 4 years or longer, or 4.5 years (e.g., 54 months) or longer, or 5 years or longer. For example, the extended period of time can be 6 months or longer. In some cases, the extended period of time is 9 months or longer. In some cases, the extended period of time is 1 year (e.g., 12 months) or longer. In some cases, the extended period of time is 1.5 years (e.g., 18 months) or longer. In some cases, the extended period of time is 2 years (e.g., 24 months) or longer. In some cases, the extended period of time is 10 years or shorter, e.g., 7.5 years or shorter, including 5 years or shorter, e.g., 2 years or shorter.

[0053] Positive control particles Embodiments of the control composition include one or more positive control particles. A "positive control particle" is a particle configured to bind to an amine-binding fluorescent particle and / or a nucleic acid-binding fluorescent particle, e.g., a bead. A "fluorescent particle" is a particle that includes 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), e.g., a fluorophore or a quantum dot. The amine-binding fluorescent particle and the nucleic acid-binding fluorescent particle can be an amine-reactive dye and a DNA intercalating dye, respectively, for flow cytometry viability assays.

[0054] As described above, flow cytometry viability assays include fluorescent amine-reactive dyes and / or DNA intercalating dyes for staining samples containing cells. In non-viable cells (i.e., dead or dying cells), the damaged cell membrane allows the dye to enter the cell and interact with cellular components, resulting in strong fluorescent labeling. Amine-reactive dyes interact with free amines inside the cell, while DNA-binding dyes penetrate into 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., live / healthy cells) does not allow the dye to enter the interior of the cell, resulting in relatively dim staining of viable cells. In some cases, a particle analyzer (e.g., a flow cytometer) is used to measure the fluorescence intensity.

[0055] In some embodiments, a control composition (e.g., a CVC composition) containing the positive control particles of the present disclosure is used to determine the compensation or spillover values for flow cytometry cell viability assays. In flow cytometry, compensation is performed to correct for fluorescence spillover, e.g., the additional detection of a fluorescence signal (e.g., the emission of a specific fluorophore) in a detection channel other than the detection channel specifically dedicated to measuring the fluorescence signal. The wavelength range of a given detection channel can be determined by the emission wavelength or wavelengths of the fluorophore to be detected (e.g., the fluorophore of an amine-reactive dye or a DNA-binding dye). To correct for fluorescence spillover, the positive control particles of the present disclosure can be used to determine the amount of fluorescence spillover produced by a given fluorescent particle (e.g., a given cell viability dye). In these cases, 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) in order to label the positive control particles. The labeled control composition (i.e., containing the labeled positive control particles) can then be introduced into a flow cytometer in order to measure its fluorescence at one or more excitation wavelengths. The fluorescence measurement values generated from the labeled control composition can then be used to calculate the spillover or compensation values for each fluorescent particle (e.g., cell viability dye) of the cell viability assay. In some cases, the spillover or compensation value can be calculated by determining the amount of fluorescence detected for each fluorescent particle in a secondary channel (i.e., a channel other than the detection channel specifically designated for measuring the given fluorescent particle). The compensation / spillover values can then be used to adjust the flow cytometry data collected from the labeled sample cells of the cell viability assay.

[0056] As described above, the positive control particles of the control composition comprise: a core; a 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. "Covalently associated" means that the aminated polymer and the nucleic acid are covalently bound directly or via a linker to the core surface. 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 the 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 relatively evenly dispersed on the core surface to form a coating layer on the core. In these cases, the positive control particles may comprise multiple layers of each of the aminated polymer and the nucleic acid, e.g., 2 or more layers each, or 3 or more layers each, or 4 or more layers each, or 5 or more layers each, or 10 or more layers each, etc.

[0057] In some cases, the aminated polymer and the nucleic acid molecules are layered alternately. In other words, each layer comprises one or more aminated polymer molecules or one or more nucleic acid molecules, and there are no two consecutive aminated polymer layers or nucleic acid layers. In other cases, the aminated polymer and the nucleic acid are uniformly mixed and dispersed on the core surface. In some cases, the positive control particles comprise a number of layers sufficient to bind (e.g., for a given cell viability assay) a certain amount of amine-reactive dye and / or DNA-binding dye such that the positive control particles are at least as bright as the cells stained with the same dyes (e.g., during a cell viability assay). In these cases, the cells may be mammalian cells, for example. In these cases, the positive control particles may comprise a number of layers sufficient to bind the same or a greater amount of amine-reactive dye and / or DNA-binding dye as non-viable cells of a particular cell viability assay (e.g., non-viable mammalian tissue cells grown under specific conditions). In some embodiments, the positive control particles may be configured to have substantially the same autofluorescence as the cells of a given flow cytometry assay.

[0058] As described above, aspects of the control particles of the present disclosure include a core. In some cases, the core of the control particle does not substantially autofluoresce when exposed to light having a wavelength in the range of, for example, from about 350 nm to about 1000 nm. "Does not substantially autofluoresce" means that when irradiated with a light beam sufficient to cause a fluorescent particle to fluoresce, the core emits less fluorescence than a cell or control particle labeled with a fluorescent particle (e.g., a cell viability dye). In other words, the core of the present disclosure is configured such that fluorescent events generated by fluorescent particles (e.g., labeled positive control particles or sample cells) can be easily and reliably distinguished from background fluorescence from the core. Thus, the fluorescence inherently emitted by the core (i.e., the autofluorescence of the core) does not interfere with accurately determining the spillover value of the fluorescent particles used in a flow cytometry assay (e.g., the spillover value of a cell viability dye in a flow cytometry cell viability assay).

[0059] In some embodiments, the control compositions of the present disclosure are configured such that when used to determine the compensation or spillover value in a flow cytometry cell viability assay, the inherent fluorescence of the positive control particles (including, for example, the inherent fluorescence of a substantially non-autofluorescent core) will not substantially contribute to fluorescence spillover. In some cases, the core comprises a low autofluorescent material that does not emit fluorescence or substantially does not emit fluorescence. In some embodiments, the core comprises a low autofluorescent material that does not emit fluorescence or emits substantially no fluorescence in a predetermined wavelength range for calculating the spillover value of the fluorescent particles (e.g., a cell viability dye in a cell viability assay) used in a flow cytometry assay of interest. In certain embodiments, the core comprises a low autofluorescent material that does not emit fluorescence or emits substantially no fluorescence when excited by ultraviolet (UV) light (e.g., from about 320 nm to 380 nm) or violet light (e.g., from about 390 nm to 420 nm).

[0060] In some embodiments, the core of the present disclosure exhibits low non-specific binding. "Low non-specific binding" means that the core exhibits low affinity (e.g., having a relatively high dissociation constant upon interaction) for unwanted biological materials (e.g., proteins, polysaccharides, nucleic acids, cells, etc.) and non-biological materials (e.g., silica, cationic polymers, plastics, etc.), such that little (if any) of the unwanted materials bind to, interact with, or hybridize with the core. In some embodiments, the core of the present disclosure exhibits low non-specific binding to fluorescent particles (e.g., stains, dyes, labeled antibodies, etc.) in flow cytometry assays and substantially does not interact with such fluorescent particles (e.g., has low non-specific binding to cell viability dyes for 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., as described herein) and the amine-reactive dye. In some embodiments, the dissociation constant of the interaction between the core and a 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., as described herein) and the DNA-binding dye. In some embodiments, the core exhibits low non-specific binding to any material used to prepare a sample for flow cytometry analysis and substantially does not interact with such any material.

[0061] The core of the present disclosure can include, but is not limited to, microparticles, beads (e.g., microbeads, magnetic beads, ion stream beads, flow cytometry beads) or microspheres. The core of the present disclosure can comprise any material that allows the core to substantially not autofluoresce (e.g., at the excitation wavelength of the fluorescent particles in the flow cytometry assay of interest) and exhibits low non-specific 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 can include polymers (e.g., plastics, polydimethylsiloxane [PDMS], polystyrene, polypropylene, agarose, gelatin, hydrogels, methylstyrene, acrylic polymers, latex, agarose, cellulose, nylon, silicone, poly[methyl methacrylate] [PMMA], hydrogels, polyacrylamide hydrogels, polysulfone, polyethyl, etc.), ceramics, glass, metals (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 a silica bead. In other embodiments, the core can be a PMMA bead or a polyacrylamide hydrogel bead.

[0062] 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 roughly match the size of the cells being analyzed, e.g., using a flow cytometry cell viability assay. In some embodiments, the core has a diameter of from about 0.1 to about 150 microns, such as from about 0.1 to 0.5 microns, or from about 0.5 to 1 micron, or from about 1 to 1.5 microns, or from about 1.5 to 5 microns, or from about 5 to 7 microns, or from about 7 to 10 microns, or from about 10 to 25 microns, or from about 25 to 50 microns, or from about 50 to 100 microns, or from 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, spherical, disk-shaped, oval, cubic, cylindrical, pyramidal, irregular, or any other suitable shape. In some embodiments, the core is solid. In other embodiments, the core is hollow.

[0063] 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 covalently bound to the core. In some cases, the fluorescent particle-binding molecules are indirectly, e.g., via a linker, covalently bound to the core. 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, an aminated polymer molecule can be covalently bound to a nucleic acid molecule, wherein the nucleic acid molecule is directly or via a linker covalently bound to the surface of the core. In some embodiments, the core can be modified to include chemical groups to facilitate conjugation of the fluorescent particle-binding molecules. Suitable modifications and linkers will be further described below. In certain embodiments, the fluorescent particle-binding molecules conjugated to the core surface are configured to bind to cell viability assay dyes (e.g., amine-reactive dyes or DNA-binding dyes) and can include the aminated polymers and / or nucleic acids discussed above and further described below. In some embodiments, molecules configured to reduce the binding of unwanted materials (e.g., unwanted biological compounds, non-biological compounds, stains, dyes, antibodies, or other fluorescent or labeled molecules) are conjugated to the surface of the core. Such molecules can include, but are not limited to, for example, polyethylene glycol (PEG) 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.

[0064] As described above, aspects of the positive control particles of the present disclosure include a core covalently associated with a molecule comprising an amine that is configured to react with an amine-reactive dye. Any molecule or combination of molecules comprising an amine (e.g., a primary amine, secondary amine, or tertiary amine) that can bind an amine-reactive dye can be used in the positive control particles. In some cases, the molecule is an aminated polymer. "Aminated polymer" refers only to a polymer having one or more amines. In certain embodiments, one or more aminated polymers of the positive control particles each comprise a plurality of amines. In these cases, the aminated polymer can be configured to prevent self-quenching of the amine-reactive dye. For example, the aminated polymer can be selected such that its structure does not contribute to self-quenching, e.g., the aminated polymer can include a spacing between each amine sufficient to prevent self-quenching of the amine-reactive dye.

[0065] In some embodiments, one or more aminated polymers of the positive control particles include cationic polymers, such as polysaccharides. In some cases, the cationic polymer includes one or more of the following: branched polyethyleneimine, polyallylamine, polylysine, or chitosan. In some embodiments, the aminated polymer includes a polypeptide, such as a protein. In these cases, the protein can include arginine and / or lysine residues. In some embodiments, the protein is histone or myelin basic protein (MBP). In some cases, the protein can be configured to bind to other fluorescent particles in addition to the amine-reactive dye. For example, the protein can be configured to bind to a specific binding member conjugated to a fluorophore, where the specific binding member can include, for example, an antibody. As used herein, the term "antibody" is used to refer to a protein composed of one or more polypeptides substantially encoded by an immunoglobulin gene that is fully or partially recognized. In some cases, the protein can be an epitope of an antigen, and the antibody containing the fluorescent particle is configured to bind to the antigen epitope. In some embodiments, the positive control particles can include two or more different aminated polymers, such as two or more different proteins. For example, the positive control particles can include 5 layers of aminated polymer molecules, where 4 layers of the aminated polymer layers include histone and the outermost aminated polymer layer includes CD protein.

[0066] In some embodiments, 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 a maximum emission wavelength in the range from 350 nm to 900 nm. For example, in some embodiments, the amine-reactive dye has a maximum emission wavelength in the range from 400 nm to 900 nm or 400 nm to 850 nm. In some embodiments, the amine-reactive dye has a maximum absorption (excitation maximum) in the range from 300 nm to 1000 nm. For example, in some embodiments, the amine-reactive dye has a maximum absorption wavelength in the range from 300 nm to 900 nm or 300 nm to 800 nm. In some embodiments, the amine-reactive dye includes BD Horizon™ reagents, such as BD Horizon™ Fixable Viability Staining (FVS) reagents. In some cases, the amine-reactive dye includes at least one dye selected from the group consisting of: BD Horizon™ Fixable Viability Stains, Biolegend Zombie™ dyes, Thermo Fischer eFluor™ dyes, Thermo Fischer LIVE / DEAD™ dyes, Proteintech Phantom dyes, and Tonbo Biosceinces Ghost Dye™.

[0067] As described above, aspects 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 a DNA-binding dye (e.g., a DNA intercalating dye) can be used for the positive control particles. In certain embodiments, one or more nucleic acids of the positive control particles have a size from 200 base pairs to 1000 base pairs. In certain embodiments, one or more nucleic acids of the positive control particles have a size from 300 base pairs to 700 base pairs, such as 400 base pairs to 600 base pairs, including for example 450 base pairs to 550 base pairs. In some embodiments, one or more nucleic acids of the positive control particles are single-stranded. In certain embodiments, one or more nucleic acids of the positive control particles are double-stranded.

[0068] In some embodiments, one or more nucleic acids of the control particles each consist essentially of DNA. In some embodiments, the DNA includes double-stranded DNA (dsDNA). In some embodiments, the DNA includes naturally occurring DNA sequences, such as DNA sequences obtained from salmon. In some embodiments, the DNA includes synthetic DNA sequences. 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 DNA intercalating dyes. For example, the nucleic acid (e.g., a dsDNA molecule) can be configured to bind to a specific binding member conjugated to a fluorophore, where the specific binding member can include, for example, a nucleic acid binding probe. In other words, the nucleic acid can include dsDNA having a sequence configured to bind to a specific gene probe (e.g., a single-stranded DNA or RNA fragment conjugated to a fluorophore). In some embodiments, the positive control particles can include two or more different nucleic acids, e.g., two or more dsDNA molecules, each molecule including a different sequence. For example, the positive control particles can include 5 layers of nucleic acid molecules, where 4 nucleic acid layers include dsDNA sequences obtained from salmon, and the outermost nucleic acid layer includes a dsDNA sequence substantially identical to all or part of the gene of interest.

[0069] In some embodiments, one or more nucleic acids are configured to bind to a DNA-binding dye that is free-flowing in solution. In some embodiments, the DNA-binding dye has a maximum emission wavelength from 350 nm to 900 nm. For example, in some embodiments, the DNA-binding dye has a maximum emission wavelength from 400 nm to 900 nm or 400 nm to 850 nm. In some embodiments, the DNA-binding dye has a maximum absorption (excitation maximum) from 300 nm to 1000 nm. For example, in some embodiments, the DNA-binding dye has a maximum absorption wavelength from 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-diamidino-2-phenylindole (DAPI). In some cases, the DNA-binding dye includes at least one dye selected from the following: 7-AAD, DAPI, propidium iodide, Hoechst dyes, ethidium bromide, LDS 751, Thermo Fischer Sytox™ dyes, Thermo Fischer T-PRO™ dyes, Thermo Fischer TOTO™ dyes, Thermo Fischer YO-PRO™ dyes, Biolegend Helix-NP™ dyes, Biotium RedDot™ dyes, and Biostatus Limited DRAQ™ dyes.

[0070] As described above, aspects of the positive control particles of the present disclosure include a core covalently associated with a nucleic acid and an aminated polymer (e.g., as described herein). In some embodiments, the nucleic acid and / or the aminated polymer are directly covalently bound to the surface of the core. In some cases, the nucleic acid and / or the aminated polymer are indirectly covalently bound to the surface of the core, e.g., through 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, an aminated polymer molecule can be covalently bound to a nucleic acid molecule, where the nucleic acid molecule is directly or through a linker covalently bound to the surface of the core.

[0071] The linker employed can include, for example, one or more reactive groups configured to stably associate two components of the positive control particle together. For example, suitable linkers can include, but are not limited to, for example, carboxyl-to-amine reactive groups, amine reactive groups, thiol reactive groups, aldehyde reactive groups, photoreactive groups, or hydroxyl reactive groups. In some embodiments, the linker employs reactive ligation chemistries, such as where the reactive linker pairs (e.g., provided by the linker and chemical groups such as the core, aminated polymer, or nucleic acid described herein) include, but are not limited to: maleimide / thiol; thiol / thiol; pyridyldithiol / thiol; succinimidyl iodoacetate / thiol; N-succinimidyl ester (NHS ester), sulfonated dichlorophenol ester (SDP ester), or pentafluorophenyl ester (PFP ester) / amine; disuccinimidyl ester / amine; imidoester / amine; hydrazine or amine / aldehyde, dialdehyde, or benzaldehyde; isocyanate / hydroxyl or amine; carbohydrate-periodate / hydrazine or amine; diazine / aryl azide chemistry; pyridyldithiol / aryl azide chemistry; alkyne / azide; carboxyl carbodiimide / amine; amine / sulfo-SMCC (sulfo-succinimidyl 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 / hydrazinonicotinamide. In certain embodiments, the linker employs cycloaddition reactions, such as [1+2]-cycloaddition, [2+2]-cycloaddition, [3+2]-cycloaddition, [2+4]-cycloaddition, [4+6]-cycloaddition, or chelation reactions, including linkers that undergo 1,3-dipolar cycloaddition (e.g., azide-alkyne Huisgen cycloaddition), Diels-Alder reaction, inverse electron demand Diels-Alder cycloaddition, ene reaction, or [2+2] photochemical cycloaddition reaction. In some embodiments, the linker can include an alkyl chain, an alkoxy chain, an alkenyl chain, or an alkynyl chain, where the number of carbon atoms in the chain can vary, in some cases ranging from 2 to 25, such as 5 to 20, where one or more carbon atoms are replaced with, for example, NH or CH3-N as reactive functionalities for covalent bonding.

[0072] In some embodiments, the aminated polymers and nucleic acids are layered on the core. In some embodiments, multiple aminated polymer layers of the coating layer and / or the nucleic acid layer of the coating layer are covalently associated with the core. For example, the particles can include 5 or more aminated polymer layers and / or 5 or more nucleic acid layers. In some embodiments, the aminated polymer layers of the coating layer are alternated with the nucleic acid layers of the coating layer. In other words, each layer includes one or more aminated polymer molecules or one or more nucleic acid molecules, and there are no two consecutive aminated polymer layers or nucleic acid layers. In these cases, the positive control particles can include multiple layers of aminated polymers and nucleic acids, for example, 2 or more layers each, or 3 or more layers, or 4 or more layers, or 5 or more layers, or 10 or more layers, etc. In other cases, the aminated polymers and nucleic acids are uniformly mixed and dispersed on the surface of the core. In some cases, the positive control particles include a number of layers sufficient to bind (e.g., for a given cell viability assay) a certain amount of amine-reactive dye and / or DNA-binding dye such that the positive control particles are at least as bright as the cells stained with the same dye (e.g., during a cell viability assay). In these cases, the positive control particles can include a number of layers sufficient to bind the same or a greater amount of amine-reactive dye and / or DNA-binding dye as non-viable cells (e.g., non-viable mammalian tissue cells grown under specific conditions) in a particular cell viability assay.

[0073] Negative control particles In certain embodiments, the control compositions of the present disclosure can include one or more negative control particles. A "negative control particle" refers to a particle that is configured to have a low affinity (e.g., a high dissociation constant upon interaction) for amine-binding fluorescent particles and / or nucleic acid-binding fluorescent particles, such as beads, to which the corresponding positive control particles (e.g., as described above) are configured to bind relative 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 in 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 particles are beads.

[0074] In some cases, the negative control particles include a core. In some cases, the core of the negative control particles can be substantially the same as the core of the positive control particles. For example, the core of the negative control particles can include the same material, 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 can be substantially the same as the core of the positive control particles. In some embodiments, the core of the negative control particles comprises a material having low autofluorescence and / or low non-specific binding. In some embodiments, for example, when considered as a whole, the core of the negative control particles does not substantially autofluoresce and / or exhibits low non-specific binding.

[0075] In certain embodiments, the negative control particles do not contain an aminated polymer or nucleic acid (i.e., do not contain any of the aminated polymer molecules or any nucleic acid molecules described herein). In some cases, the negative control particles contain a polymer having substantially the same autofluorescence as the aminated polymer of the positive control (e.g., as described above), wherein the negative control polymer does not react or bind to an amine-reactive dye. For example, the negative control particles can contain a protein having 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 having substantially the same autofluorescence as the nucleic acid of the positive control (e.g., as described above), wherein the negative control nucleic acid does not react or bind to a DNA-binding dye. For example, the negative control particles can contain a nucleic acid substantially devoid of G-C base pairs, and the DNA-binding dye can 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 can be PNA, while the positive control nucleic acid can be DNA.

[0076] As described above, in some embodiments, the negative control particles include a core but do not contain a polymer or nucleic acid. In these cases, the core of the negative control particles can be conjugated with a molecule to consume surface groups that can react with an amine-reactive dye or a DNA-binding dye. Such molecules can include, but are not limited to, for example, polyethylene glycol (PEG) of various molecular weights and branching 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 non-specific binding polymer layer or coating layer, such as a silane layer or a hydrophilic polymer (e.g., PEG, poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), poly(carboxybetaine acrylamide) (PCBAA), etc.) layer, can be deposited on the surface of the core of the negative control particles.

[0077] Figure 1Shows two types of viability staining mechanisms according to embodiments of the present invention. At the top, two cells are depicted before incubation with the cell viability dye. The live cell with an intact cell membrane (i.e., a live and healthy cell) is shown on the left, while the non-live cell with a damaged cell membrane (i.e., a dead or dying cell) is shown on the right. Then both the live and non-live cells are incubated with an amine-reactive dye (left middle) and a DNA intercalating dye (right middle) to distinguish between live and non-live cells. At the bottom, the live and non-live cells after incubation with the amine-reactive dye or the DNA intercalating dye are shown. For the amine-reactive dye-based assay (bottom left), the amine-reactive dye binds to the amines on the intact cell membrane proteins of the live cells but cannot enter the live cells to bind to the free amines inside the cells. In contrast, for non-live cells, the amine-reactive dye binds to both the amines of the damaged cell membrane and the amines inside the cells, resulting in a higher fluorescence intensity of the amine-reactive dye for non-live cells. For the DNA intercalating dye-based assay (bottom right), the DNA intercalating dye cannot enter the live cells to bind to the dsDNA in the cell nucleus. In contrast, for non-live cells, the DNA intercalating dye penetrates all the way into the cell nucleus to directly bind to the dsDNA molecules, resulting in a higher fluorescence intensity of the DNA intercalating dye for non-live cells. Thus, both the amine-reactive dye-based assay and the DNA intercalating dye-based assay are capable of distinguishing between live and non-live cells.

[0078] Figure 2 Provides an illustration of a positive CVC bead according to embodiments of the present invention. The positive CVC bead 200 includes a core 201 consisting essentially of silica, a DNA molecule coating layer 202 configured to bind to a DNA-binding dye, and an aminated polymer molecule coating layer 203 configured to bind to an amine-reactive dye. The DNA coating layer 202 includes a plurality of dsDNA sequences, each having a length of about 500 base pairs, which are relatively uniformly distributed over the entire surface of the core 201. The DNA coating layer 202 is covalently attached to the surface of the core 201 either directly or through a linker, and the aminated polymer coating layer 203 is covalently associated with the core 201 through a covalent bond formed with the DNA coating layer 202. The aminated polymer coating layer 203 includes a plurality of amine-rich histones, which are relatively uniformly distributed over the DNA coating layer 202.

[0079] For ease of illustration, a dsDNA molecule 204 for binding a DNA-binding dye and an amine group 205 for binding an amine-reactive dye are depicted on the exterior of the particle. The positioning of the dsDNA molecules of the DNA coating layer 202 and the histones of the aminated polymer coating layer 203 allows freely diffusing dyes in the liquid solution to come close enough for the dsDNA molecules and histones (and their amines) to interact with the freely diffusing dyes. This enables the amines of the aminated polymer coating layer 203 (e.g., 205) to bind to the amine-reactive dye of the liquid solution and the dsDNA molecules of the DNA coating layer 202 (e.g., 204) to bind to the DNA-binding dye of the liquid solution.

[0080] Manufacturing method Aspects of the present disclosure also include methods for preparing (i.e., synthesizing or generating) control particles as described herein. In certain embodiments, the preparation method can be used to generate positive control particles. In certain embodiments, the preparation method can be used to generate negative control particles. In some embodiments, one or more steps of the method can be used to generate both positive 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 generate, for example, positive control particles.

[0081] In certain embodiments, the surface of the core comprises functional groups such as amide, maleimide, or thiol groups. In some embodiments, the method further includes surface-functionalizing the core with a functional group. For example, the obtained core can comprise silica, and the functionalization can include amination. In some embodiments, the method further includes modifying the functional group of the core with a reagent, for example, to comprise a reactive functional group. For example, a thiolation reagent such as 2-iminothiolane can be used to modify an aminated silica core to comprise a thiol group. In some embodiments, the method further includes making the core positively charged, for example, when the core is covalently bound to a nucleic acid molecule (before association with the aminated polymer described herein). In some embodiments, the method further includes making the core negatively charged, for example, when the core is covalently bound to an aminated polymer (before association with the nucleic acid described herein).

[0082] In certain embodiments, the nucleic acid comprises deoxyribonucleic acid (DNA), such as double-stranded DNA (dsDNA). In some embodiments, the particle synthesis method further comprises modifying the DNA to include functional groups that react with the functional groups of the core and / or the functional groups of the aminated polymer. For example, the core and / or the aminated polymer can be modified to include thiol groups, and the DNA can be modified to include maleimide groups. In some embodiments, the particle synthesis method further comprises modifying the DNA to include functional groups that are reactive with the reactive chemistry of the docking linker (i.e., where 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 the functional groups of the DNA with the reactive functional groups 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 the functional groups of the DNA with the functional groups of the aminated polymer and / or a linker associated with the aminated polymer. In some embodiments, the 5' end and / or the 3' end of the DNA is functionalized. In some embodiments, the DNA is dsDNA, and both strands of the dsDNA are functionalized. In some embodiments, the dsDNA is covalently bound to the core by reacting the functional groups of the first strand of the dsDNA with the reactive functional groups of the core (or its linker), and the dsDNA is covalently bound to the aminated polymer by reacting the functional groups of the second strand of the dsDNA with the functional groups of the aminated polymer (or its linker). In some embodiments, both strands of the dsDNA are covalently bound to the aminated polymer.

[0083] In certain embodiments, the particle synthesis method further comprises modifying the aminated polymer to include functional groups that react with the functional groups of the core and / or the nucleic acid. For example, the core and / or the nucleic acid can be modified to include maleimide groups, and the aminated polymer can be modified to include thiol groups. In some embodiments, the particle synthesis method further comprises modifying the aminated polymer to include functional groups that react with the reactive chemistry of the linker (i.e., where the linker is further configured to bind to the core and / or the nucleic acid). In some embodiments, the aminated polymer is covalently bound to the core by reacting the functional groups of the aminated polymer with the reactive functional groups of the core and / or the linker associated with the core. In some embodiments, the aminated polymer is covalently bound to the core by reacting the functional groups of the aminated polymer with the functional groups of the nucleic acid and / or the 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 using, for example, 2-iminothiolane. In some embodiments, the aminated polymer is covalently bound to the core by reacting a first functional group of the aminated polymer with the reactive functional group of the core (or its linker), and the aminated polymer is covalently bound to the nucleic acid by reacting a second functional group of the aminated polymer with the functional group of the nucleic acid (or its linker). In some embodiments, two or more functional groups of the aminated polymer are covalently bound to the nucleic acid.

[0084] In certain embodiments, the aminated polymer and the nucleic acid are covalently associated with the core by alternately depositing coating layers of the aminated polymer and the nucleic acid. For example, the nucleic acid can include dsDNA and can be modified to include maleimide groups at the 5' end of each strand of the dsDNA, the aminated polymer can include a protein and can be modified to include two or more thiol groups, and the core can include a plurality of thiol groups (e.g., after modification). Then, the deposition can 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, and so on. In some embodiments, 5 or more rounds of deposition are performed such that the control particles include 5 or more nucleic acid layers and 5 or more aminated polymer layers. In some embodiments, each round of deposition includes covalently associating a plurality of nucleic acids and a plurality of aminated polymers to the core.

[0085] 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 a plurality of amine-reactive dyes and / or DNA-binding dyes to produce 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 as described above) to the amines of the negative control particles. In some embodiments, the negative control particles do not contain an aminated polymer or nucleic acid. In some embodiments, synthesizing 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 dye from reacting with the core.

[0086] Figure 3 Shows a synthesis according to an embodiment of the present invention Figure 2 method of positive control beads. In step 300, an aminated silica core is obtained, for example, by producing silica beads and performing surface functionalization to stably associate amines to the surface of the silica beads. In step 310, the amines of the silica beads are modified with a thiolation reagent 311 (i.e., 2-iminothiolane) such that the beads contain thiol groups. The thiolated silica beads are then made positively charged to obtain a thiolated, positively charged core 312.

[0087] In step 320, a dsDNA molecule is produced using a DNA sequence (e.g., obtained from salmon). The dsDNA molecule is then modified to contain a maleimide functional group that reacts with the thiol groups of the core at the 5' or 3' ends of the individual strands of the dsDNA molecule. A plurality of maleimide-functionalized dsDNA molecules 321 are then covalently bound to the thiolated, positively charged core to produce a silica core with a single dsDNA molecule 322 coating layer.

[0088] In step 330, histone 331 is obtained, for example by purifying a lysate of mammalian cells. The histone is then modified with a thiolation reagent 332 (i.e., 2-iminothiolane) such that the protein contains thiol groups. Multiple thiolated histones 333 are then covalently attached to the remaining maleimide groups of multiple dsDNA molecules bound to a silica core so as to produce a silica core having a single dsDNA molecule coating layer and a single histone molecule layer 334. In step 340, step 320 is repeated to attach additional dsDNA coating layers to the remaining thiol groups of the multiple histones of the outermost histone layer, and step 330 is repeated to attach additional histone coating layers to the remaining maleimide groups of the multiple dsDNA molecules of the outermost histone layer until a positive control bead having 5 amine-functionalized polymer (i.e., histone) coating layers [and 5 nucleic acid (i.e., dsDNA) coating layers 341 is produced.

[0089] Methods of Use Aspects of the present disclosure also include methods of using the subject control compositions. As described above, the control compositions can include multiple positive control particles, multiple negative control particles, or multiple positive and negative control particles. Accordingly, methods of using the control compositions can include using the compositions to verify the functionality or performance of a particle analysis system (e.g., a flow cytometry system), protocol, and reagents for use in flow cytometry assays. In some embodiments, the method includes using the control compositions to interpret flow cytometry data. In such cases, the method can include using the control compositions for fluorescence compensation of flow cytometry data.

[0090] In some embodiments, the control compositions are used for fluorescence compensation, e.g., to correct 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 compositions are used to remove the signal of a given fluorescent particle from all detectors or channels of a particle analyzer (e.g., a flow cytometer) other than the channel / detector dedicated to measuring the fluorescent particle. In some cases, fluorescence compensation can be performed for flow cytometry cell viability assays so as to, for example, distinguish live cell populations from dead cell populations in flow cytometry data. In some embodiments, an amine-reactive dye is used for flow cytometry cell viability assays to distinguish live cells from dead cells. In certain embodiments, a DNA-binding dye (e.g., a DNA intercalating dye) is used for flow cytometry cell viability assays to distinguish live cells from dead cells.

[0091] In some embodiments, a control composition comprising the positive control particles of the present disclosure is used to determine the compensation or spillover values for a flow cytometry cell viability assay. In some cases, the wavelength range for detection by a given detection channel configuration can be determined by the emission wavelength or wavelengths of the fluorophore(s) to be detected (e.g., the fluorophore of an amine-reactive dye or a DNA-binding dye). To correct for fluorescence spillover, the positive control particles of the present disclosure can be used to determine the amount of fluorescence spillover produced by a given fluorescent particle (e.g., a given cell viability dye). In these cases, 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) in order to label the positive control particles. The labeled control composition (i.e., comprising the labeled positive control particles) can then be introduced into a flow cytometer in order to measure its fluorescence at one or more excitation wavelengths. The fluorescence measurements from the labeled control composition can then be used to calculate the spillover or compensation values for each fluorescent particle (e.g., cell viability dye) of the cell viability assay. In some cases, the spillover or compensation values can be calculated by determining the amount of fluorescence detected for each fluorescent particle in a secondary channel (i.e., a channel other than the detection channel dedicated to measuring the given fluorescent particle). The compensation / spillover values can then be used to adjust the flow cytometry data collected from the labeled sample cells of the cell viability assay. In some embodiments, the labeled control composition comprises negative control particles, e.g., having substantially the same autofluorescence as the positive control particles and / or the cells of a given flow cytometry assay (e.g., cell viability assay). In these cases, the fluorescence measurements from the negative control particles can be used to adjust or modify the fluorescence measurements from the positive control particles prior to calculating the compensation / spillover values.

[0092] In certain embodiments, fluorescence minus one (FMO) controls can be performed using the subject control compositions (e.g., the negative and positive control particles of the present invention). For example, a flow cytometry assay can include three or more fluorescent particles: 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 comprises positive control particles that are configured to bind to each fluorescent particle of the assay (except for one fluorescent particle of each fluorescent particle). The control composition can then be labeled (i.e., by incubating with all of the dyes of the assay), and the labeled control composition can then be introduced into a flow cytometer in order to measure its fluorescence at one or more excitation wavelengths. The fluorescence measurements from the labeled control composition can then be used to calculate the spillover or compensation values for each fluorescent particle (e.g., cell viability dye) of the cell viability assay.

[0093] Sample analysis As described above, the subject control compositions can be used to analyze cell samples, for example, by performing a flow cytometry viability assay to determine the live and non-live cells in the sample. The cells that may be present in the sample include eukaryotic cells (e.g., mammalian cells) and / or prokaryotic cells (e.g., bacterial cells 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., bacteria, archaea) cell cultures, environmental samples containing prokaryotic and / or eukaryotic (e.g., mammalian, protist, fungal, etc.) cells, eukaryotic cell cultures (e.g., cultures of established cell lines, cultures of known or purchased cell lines, cultures of immortalized cell lines, cultures of primary cells, cultures of laboratory yeast, etc.), tissue cultures, etc. In vitro sources can further include organic tissues, including both healthy and diseased tissues (e.g., cancerous, malignant, necrotic, etc.), such as sections of skin, respiratory system, gastrointestinal tract, cardiovascular system, urogenital tract, tumors, organs, etc. In other cases, the cells of the cell sample can be obtained from fluids produced by an organism, such as blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and / or semen.

[0094] In certain embodiments, the sample source is a "mammal", a term widely used to describe organisms in the class Mammalia, including the order Carnivora (e.g., dogs and cats), the order Rodentia (e.g., mice, guinea pigs, and rats), and the order Primates (e.g., humans, chimpanzees, and monkeys). In some cases, the subject is a human. These methods can be applied to samples obtained from human subjects of both genders and any developmental stage (i.e., neonate, infant, juvenile, adolescent, adult), where in certain embodiments, the human subject is a juvenile, adolescent, or adult. While the present invention can be applied to samples from human subjects, it should be understood that these methods can also be performed on samples from other animal subjects (i.e., "non-human subjects"), such as, but not limited to, birds, mice, rats, dogs, cats, livestock, and horses.

[0095] In certain embodiments, the control compositions used in the methods of the present invention can be used in flow cytometry protocols (e.g., for analyzing samples, such as the samples described above). When practicing such methods, the sample and the control composition are irradiated with light from a light source (e.g., in the flow stream of a flow cytometer). In some embodiments, the light source is a broadband light source that emits light having a wide wavelength range, e.g., spanning 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 including spanning 500 nm or greater. For example, a suitable broadband light source emits light having a wavelength of 200 nm to 1500 nm. Another example of a suitable broadband light source includes a light source that emits light having a wavelength of 400 nm to 1000 nm. When the method includes irradiation with a broadband light source, the broadband light source protocols of interest can include, but are not limited to, halogen lamps, deuterium arc lamps, xenon arc lamps, stable fiber-coupled broadband light sources, broadband LEDs having a continuous spectrum, superluminescent light-emitting diodes, semiconductor light-emitting diodes, broadband LED white light sources, multi-LED integrated white light sources, and other broadband light sources or any combination thereof.

[0096] In other embodiments, the methods of the embodiments of the present invention include irradiation with a narrowband light source that emits a specific wavelength or a narrow wavelength range, e.g., irradiation with a light source that emits light within a narrow wavelength range, such as a range of 50 nm or less, e.g., 40 nm or less, e.g., 30 nm or less, e.g., 25 nm or less, e.g., 20 nm or less, e.g., 15 nm or less, e.g., 10 nm or less, e.g., 5 nm or less, and including a light source that emits light of a specific wavelength (i.e., monochromatic light). When the method includes irradiation with a narrowband light source, the narrowband light source protocols of interest can include, but are not limited to, narrow wavelength LEDs, laser diodes, or a broadband light source coupled with one or more optical bandpass filters, diffraction gratings, monochromators, or any combination thereof.

[0097] In certain embodiments, the method includes irradiating a sample and a control composition with one or more lasers. As described above, the type and number of lasers will vary depending on the sample and control composition and the light desired to be collected, and can be gas lasers such as helium-neon lasers, argon lasers, krypton lasers, xenon lasers, nitrogen lasers, CO2 lasers, CO lasers, argon fluoride (ArF) excimer lasers, krypton fluoride (KrF) excimer lasers, xenon chloride (XeCl) excimer lasers, or xenon fluoride (XeF) excimer lasers or combinations thereof. In other cases, the method includes irradiating a flowing stream with a dye laser (such as a stilbene, coumarin, or rhodamine laser). In other cases, the method includes irradiating a flowing stream with a metal vapor laser (such as a helium cadmium (HeCd) laser, helium mercury (HeHg) laser, helium selenium (HeSe) laser, helium silver (HeAg) laser, strontium laser, neon copper (NeCu) laser, copper laser, or gold laser and combinations thereof). In other cases, the method includes irradiating a flowing stream with a solid-state laser (such as a ruby laser, Nd:YAG laser, NdCrYAG laser, Er:YAG laser, Nd:YLF laser, Nd:YVO4 laser, Nd:YCa4O(BO3)3 laser, Nd:YCOB laser, titanium sapphire laser, thulium YAG laser, ytterbium YAG laser, ytterbium2O3 laser, or cerium-doped laser and combinations thereof).

[0098] The sample and control composition can be irradiated with one or more of the above-described light sources, such as 2 or more light sources, such as 3 or more light sources, such as 4 or more light sources, such as 5 or more light sources, and including 10 or more light sources. The light sources can include a combination of any type of light source. For example, in some embodiments, the method includes irradiating a sample and a control composition in a flowing 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).

[0099] The sample and the control composition can be irradiated with wavelengths in the range of 200 nm to 1500 nm (such as 250 nm to 1250 nm, such as 300 nm to 1000 nm, such as 350 nm to 900 nm, and including 400 nm to 800 nm). For example, when the light source is a broadband light source, the sample and the control composition can be irradiated with wavelengths of 200 nm to 900 nm. In other cases, when the light source includes a plurality of narrowband light sources, the sample and the control composition can be irradiated with specific wavelengths in the range of 200 nm to 900 nm. For example, the light source can be a plurality of narrowband LEDs (1 nm – 25 nm), and each narrowband LED independently emits light with a wavelength range of 200 nm to 900 nm. In other embodiments, the narrowband light source includes one or more lasers (such as a laser array), and the sample and the control composition can be irradiated with specific wavelengths in the range of 200 nm to 700 nm, for example, using a laser array having gas lasers, excimer lasers, dye lasers, metal vapor lasers, and solid-state lasers as described above.

[0100] When more than one light source is used, the sample and the control composition can be irradiated with the light source simultaneously, sequentially, or in a combination of both. For example, the sample and the control composition can be irradiated simultaneously with each light source. In other embodiments, the flowing stream is irradiated sequentially with each light source. When the sample and the control composition are irradiated sequentially with more than one light source, the irradiation time of each light source on the sample and the control composition can independently be 0.001 microseconds or longer, such as 0.01 microseconds or longer, such as 0.1 microseconds or longer, such as 1 microseconds or longer, such as 5 microseconds or longer, such as 10 microseconds or longer, such as 30 microseconds or longer, and including 60 microseconds or longer. For example, the method can include irradiating the sample and the control composition with a light source (such as a laser), and the irradiation duration ranges from 0.001 microseconds to 100 microseconds, such as 0.01 microseconds to 75 microseconds, such as 0.1 microseconds to 50 microseconds, such as 1 microseconds to 25 microseconds, and including 5 microseconds to 10 microseconds. In embodiments where the sample and the control composition are irradiated sequentially with two or more light sources, the irradiation duration of each light source on the sample and the control composition can be the same or different.

[0101] The time period between irradiations of each light source can also vary as needed, with independent intervals of 0.001 microseconds or longer delays, such as 0.01 microseconds or longer, such as 0.1 microseconds or longer, such as 1 microseconds or longer, such as 5 microseconds or longer, such as 10 microseconds or longer, such as 15 microseconds or longer, such as 30 microseconds or longer, and including 60 microseconds or longer. For example, the time period between irradiations of each light source can range from 0.001 microseconds to 60 microseconds, such as 0.01 microseconds to 50 microseconds, such as 0.1 microseconds to 35 microseconds, such as 1 microseconds to 25 microseconds, and including 5 microseconds to 10 microseconds. In some embodiments, the time period between irradiations of each light source is 10 microseconds. In embodiments where the sample and the control composition are irradiated by more than two (i.e., 3 or more) light sources in sequence, the delays between irradiations of each light source can be the same or different.

[0102] The sample and the control composition can be irradiated continuously or at non - continuous intervals. In some cases, the method includes continuously irradiating the sample and the control composition with a light source. In other cases, the sample and the control composition are irradiated with a light source at non - continuous intervals, such as at intervals of every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and including every 1000 milliseconds or some other interval.

[0103] Depending on the light source, the sample and the control composition can be irradiated from different distances, such as 0.01 mm or farther, such as 0.05 mm or farther, such as 0.1 mm or farther, such as 0.5 mm or farther, such as 1 mm or farther, such as 2.5 mm or farther, such as 5 mm or farther, such as 10 mm or farther, such as 15 mm or farther, such as 25 mm or farther, and including 50 mm or farther. Additionally, the angle of irradiation can also vary, ranging from 10° to 90°, such as from 15° to 85°, such as from 20° to 80°, such as from 25° to 75°, and including from 30° to 60°, such as at a 90° angle.

[0104] In some cases, a sample and a control composition can be irradiated with frequency-shifted light deflected at multiple angles, and cells in a flowing stream can be imaged by fluorescence imaging using frequency-tagged emission (FIRE) to produce a frequency-encoded image, such as those described in Diebold, et al. Nature Photonics Vol. 7(10); 806-810 (2013) and U.S. Patent Nos. 9,423,353, 9,784,661, 9,983,132, 10,006,852, 10,078,045, 10,036,699, 10,222,316, 10,288,546, 10,324,019, 10,408,758, 10,451,538, 10,620,111 and U.S. Patent Publication Nos. 2017 / 0133857, 2017 / 0328826, 2017 / 0350803, 2018 / 0275042, 2019 / 0376895 and 2019 / 0376894, the disclosures of which are incorporated herein by reference.

[0105] In an embodiment, light from the irradiated sample and the irradiated control composition is transmitted to a light detection system and measured by one or more photodetectors. When practicing the subject methods, light from the sample and the control composition is transmitted to three or more wavelength separators, each wavelength separator being 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 sub-spectral range to a photodetector.

[0106] The light can be measured continuously or at discontinuous intervals with the light detection system. In some cases, the method includes continuously measuring the light. In other cases, the light is measured at discontinuous intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds and including every 1000 milliseconds or some other interval.

[0107] During the subject methods, the collected light can be measured one or more times, such as 2 or more times, such as 3 or more times, such as 5 or more times and including 10 or more times. In certain embodiments, the light is measured propagating 2 or more times and in some cases the data is averaged.

[0108] In some embodiments, the method includes conditioning light prior to detecting the light with the subject light detection system. For example, light from the sample and the control composition can pass through one or more lenses, mirrors, pinholes, slits, gratings, light refractors, and any combination thereof. In some cases, the collected light passes through one or more focusing lenses, for example to reduce the profile of the light of the light detection system or the optical collection system as described above. In other cases, the emitted light from the sample and the emitted light from the control composition pass through one or more collimators to reduce the beam divergence transmitted to the light detection system.

[0109] System Aspects of the invention also include a system configured to perform the methods described above. Systems of interest include a processor configured to display results according to embodiments of the invention. In an embodiment, the subject processor operates in conjunction with programmable logic, which can be implemented in hardware, software, firmware, or any combination thereof to display results. In some such embodiments, the system includes a display configured to depict a visualization. Any suitable display can be employed. The subject display can include, but is not limited to, a display, a tablet computer, a smart phone, or other electronic device configured to present a graphical interface. In this section, the term "sample" can refer to the cell samples and control compositions of the present disclosure, or only to the cell samples depending on the context.

[0110] The subject programmable logic can be implemented in a variety of devices, such as a specially programmed event processing computer, a wireless communication device, an integrated circuit device, and the like. In some embodiments, the programmable logic can be executed by a specially programmed processor, which can include one or more processors, such as one or more digital signal processors (DSPs), a configurable microprocessor, an application specific integrated circuit (ASIC), a field programmable gate array (FPGA), or other equivalent integrated or discrete logic circuits. Combinations of computing devices, such as a combination of a DSP and a microprocessor, multiple microprocessors, a combination of one or more microprocessors and a DSP core, or any other such configuration in at least part of the data connection, can implement one or more of the features described herein.

[0111] In certain cases, the system is or includes a particle analyzer. Particle analyzers of interest can include a flow cell for transporting particles in a flowing stream, a light source for irradiating the particles in the flowing stream at a probing 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.

[0112] As described herein, a "flow cell" is described in its conventional sense as referring to a component that includes a flow channel, such as a cuvette, that has a liquid flow stream for transporting particles in a sheath fluid. Cuvettes of interest include containers having a channel therethrough. The flow stream can include a liquid sample injected from a sample tube. Flow cells of interest include optically accessible flow channels. In some cases, the flow cell includes a transparent material (e.g., quartz) that allows light to pass through. In some embodiments, the flow cell is an air flow cell in which light interrogation of the particles occurs outside of the flow cell (i.e., in free space).

[0113] In some cases, the flow stream is configured to irradiate with light from a light source at an interrogation point. The flow stream configured for the flow channel can include a liquid sample injected from a sample tube. In certain embodiments, the flow stream can include a narrow, fast flowing liquid stream that is arranged such that linearly separated particles transported therein are separated from one another in a single file manner. The "interrogation point" discussed herein refers to a region within the flow cell in which particles are irradiated with light from a light source, e.g., for analysis. The size of the interrogation point can vary as needed. For example, when 0 μm represents the optical axis of the light emitted by the light source, the interrogation point can range from -100 μm to 100 μm, such as -50 μm to 50 μm, such as -25 μm to 40 μm, and includes -15 μm to 30 μm.

[0114] After irradiating the particles in the flow cell, particle-modulated light can be observed. "Particle-modulated light" refers to the light received from the particles in the flow stream after irradiating the particles with light from a light source. In some cases, the particle-modulated light is side-scattered light. As described herein, side-scattered light refers to light that is refracted and reflected from the surface and internal structure of the particles. In additional embodiments, the particle-modulated light includes forward-scattered light (i.e., light that travels primarily forward through or around the particles). In other cases, the particle-modulated light includes fluorescence (i.e., light emitted by a fluorescent dye after irradiation with light at an excitation wavelength).

[0115] As described above, aspects of the present invention also include a light source configured to irradiate particles passing through the flow cell at the interrogation point. Any convenient light source can be used as the light source described herein. In some embodiments, the light source is a laser. In an embodiment, the laser can be any convenient laser, such as a continuous wave laser. For example, the laser can be a diode laser, such as an ultraviolet diode laser, a visible diode laser, and a near-infrared diode laser. In other embodiments, the laser can be a helium-neon (HeNe) laser. In some cases, the laser is a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon fluoride (ArF) excimer laser, a krypton fluoride (KrF) excimer laser, a xenon chloride (XeCl) excimer laser, or a xenon fluoride (XeF) excimer laser or a combination thereof. In other cases, the subject flow cytometer includes a dye laser, such as a stilbene, coumarin, or rhodamine laser. In other cases, the lasers of interest include metal vapor lasers, such as a helium cadmium (HeCd) laser, a helium mercury (HeHg) laser, a helium selenium (HeSe) laser, a helium silver (HeAg) laser, a strontium laser, a neon copper (NeCu) laser, a copper laser, or a gold laser and combinations thereof. In other cases, the subject flow cytometer includes a solid-state laser, such as a ruby laser, an Nd:YAG laser, an NdCrYAG laser, an Er:YAG laser, an Nd:YLF laser, an Nd:YVO4 laser, an Nd:YCa4O(BO3)3 laser, an Nd:YCOB laser, a titanium sapphire laser, a thulium YAG laser, a ytterbium YAG laser, a ytterbium2O3 laser, or a cerium-doped laser and combinations thereof.

[0116] The laser light source according to certain embodiments can also include one or more optical conditioning components. In certain embodiments, the optical conditioning components are located between the light source and the flow cell and can include any device capable of changing the spatial width of the irradiation or some other characteristic of the irradiation from the light source, such as the irradiation direction, wavelength, beam width, beam intensity, and focus. The optical conditioning scheme can 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 splitters, pinholes, slits, collimation schemes, and combinations thereof. In certain embodiments, the subject flow cytometer includes one or more focusing lenses. In one example, the focusing lens can be a reducing lens. In other embodiments, the subject flow cytometer includes optical fibers.

[0117] When the optical adjustment assembly is configured to move, the optical adjustment assembly can be configured to move continuously or at discrete intervals, such as in increments of 0.01 μm or greater, such as 0.05 μm or greater, such as 0.1 μm or greater, such as 0.5 μm or greater, such as 1 μm or greater, such as 10 μm or greater, such as 100 μm or greater, such as 500 μm or greater, such as 1 mm or greater, such as 5 mm or greater, such as 10 mm or greater, and including increments of 25 mm or greater.

[0118] Any displacement scheme can be employed to move the optical adjustment assembly structure, such as being coupled to a movable support stage or directly coupled to a motor-actuated translation stage, a lead screw translation assembly, a gear translation device, such as those employing a stepper motor, a servo motor, a brushless motor, a brushed DC motor, a microstep drive motor, a high-resolution stepper motor, and other types of motors.

[0119] The light source can be positioned at any suitable distance from the flow cell, such as where the light source and the flow cell are spaced 0.005 mm or greater, such as 0.01 mm or greater, such as 0.05 mm or greater, such as 0.1 mm or greater, such as 0.5 mm or greater, such as 1 mm or greater, such as 5 mm or greater, such as 10 mm or greater, such as 25 mm or greater, and including distances of 100 mm or greater. Additionally, the light source can be positioned at any suitable angle relative to the flow cell, such as at an angle in the range from 10 degrees to 90 degrees, such as from 15 degrees to 85 degrees, such as from 20 degrees to 80 degrees, such as from 25 degrees to 75 degrees and including from 30 degrees to 60 degrees, such as at an angle of 90 degrees.

[0120] In some embodiments, the light sources of interest include a plurality of lasers configured to provide lasers for non-continuous irradiation of the flowing stream, such as 2 lasers or more, such as 3 lasers or more, such as 4 lasers or more, such as 5 lasers or more, such as 10 lasers or more, and including 15 lasers or more, which are configured to provide lasers for non-continuous irradiation of the flowing stream. Depending on the optical wavelength required for irradiating the flowing stream, each laser can have a specific wavelength that varies from 200 nm to 1500 nm, such as from 250 nm to 1250 nm, such as from 300 nm to 1000 nm, such as from 350 nm to 900 nm and including from 400 nm to 800 nm. In certain embodiments, the lasers of interest can include one or more of a 405 nm laser, a 488 nm laser, a 561 nm laser, and a 635 nm laser.

[0121] As described above, the particle analyzer of interest can further include one or more particle-modulated light detectors for detecting particle-modulated light intensity data. In some embodiments, the particle-modulated light detectors include one or more forward-scattering light detectors configured to detect forward-scattered light. For example, the subject particle analyzer can include 1 forward-scattering light detector or multiple forward-scattering light detectors, such as 2 or more, such as 3 or more, such as 4 or more, and including 5 or more. In certain embodiments, the particle analyzer includes 1 forward-scattering light detector. In other embodiments, the particle analyzer includes 2 forward-scattering light detectors.

[0122] Any convenient detector for detecting the collected light can be used for the forward-scattering light 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 (CCD), intensified charge-coupled devices (ICCD), light-emitting diodes, photon counters, bolometers, thermoelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes (PMT), phototransistors, quantum dot photoconductors or photodiodes and combinations thereof, and other detectors. In certain embodiments, the collected light is measured using 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, such as a photomultiplier tube having an effective detection surface area for each region, the surface area ranging from 0.01 cm 2 to 10 cm 2 e.g., 0.05 cm 2 to 9 cm 2 e.g., 0.1 cm 2 to 8 cm 2 e.g., 0.5 cm 2 to 7 cm 2 and including 1 cm 2 to 5 cm 2 .

[0123] In an embodiment, the forward-scattering light detector is configured to measure the light continuously or at non-continuous intervals. In some cases, the detector of interest is configured to continuously measure the collected light. In other cases, the detector of interest is configured to make measurements at non-continuous intervals, such as measuring the light every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and including every 1000 milliseconds or some other interval.

[0124] In additional embodiments, one or more particle-modulated light detectors can include one or more side-scattering light detectors for detecting light at side-scattering wavelengths (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-scattering light detector. In other embodiments, the particle analyzer includes multiple side-scattering light detectors, such as two or more, three or more, four or more, and including five or more.

[0125] Any convenient detector for detecting the collected light can be used for the side-scattering light 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 (CCD), intensified charge-coupled devices (ICCD), light-emitting diodes, photon counters, bolometers, thermoelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes (PMT), phototransistors, quantum dot photoconductors or photodiodes and combinations thereof, and other detectors. In certain embodiments, the collected light is measured using a charge-coupled device (CCD), semiconductor charge-coupled device (CCD), active pixel sensor (APS), complementary metal-oxide-semiconductor (CMOS) image sensor, or N-type metal-oxide-semiconductor (NMOS) image sensor. In certain embodiments, the detector is a photomultiplier tube, such as a photomultiplier tube having an effective detection surface area for each region, the surface area ranging from 0.01 cm 2 to 10 cm 2 , such as 0.05 cm 2 to 9 cm 2 , such as 0.1 cm 2 to 8 cm 2 , such as 0.5 cm 2 to 7 cm 2 and including 1 cm 2 to 5 cm 2 .

[0126] In an embodiment, the subject particle analyzer further includes a fluorescence light detector configured to detect light at one or more fluorescence wavelengths. In other embodiments, the particle analyzer includes multiple fluorescence light detectors, such as two or more, such as three or more, such as four or more, five or more, ten or more, fifteen or more, and including twenty or more.

[0127] Any convenient detector for detecting the collected light can be used in the fluorescence 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 (CCD), intensified charge-coupled devices (ICCD), light-emitting diodes, photon counters, bolometers, thermoelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes (PMT), phototransistors, quantum dot photoconductors or photodiodes and combinations thereof, and other detectors. In certain embodiments, a charge-coupled device (CCD), semiconductor charge-coupled device (CCD), active pixel sensor (APS), complementary metal-oxide-semiconductor (CMOS) image sensor or N-type metal-oxide-semiconductor (NMOS) image sensor is used to measure the light from the collected light. In certain embodiments, the detector is a photomultiplier tube, such as a photomultiplier tube having an effective detection surface area for each region, the surface area ranging from 0.01 cm 2 to 10 cm 2 e.g., 0.05 cm 2 to 9 cm 2 e.g., 0.1 cm 2 to 8 cm 2 e.g., 0.5 cm 2 to 7 cm 2 and includes 1 cm 2 to 5 cm 2 .

[0128] When the subject particle analyzer includes a plurality of fluorescence light detectors, each fluorescence light detector can be the same, or the set of fluorescence light detectors can be a combination of different types of detectors. For example, when the subject particle analyzer includes two fluorescence light detectors, in some embodiments, the first fluorescence light detector is a CCD-type device, while the second fluorescence light detector (or imaging sensor) is a CMOS-type device. In other embodiments, the first and second fluorescence light detectors are both CCD-type devices. In other embodiments, the first and second fluorescence light detectors are both CMOS-type devices. In other embodiments, the first fluorescence light detector is a CCD-type device, while the second fluorescence light detector is a photomultiplier tube (PMT). In other embodiments, the first fluorescence light detector is a CMOS-type device, while the second fluorescence light detector is a photomultiplier tube. In other embodiments, the first and second fluorescence light detectors are both photomultiplier tubes.

[0129] In embodiments of the present disclosure, a fluorescence light detector of interest is configured to measure collected light at one or more wavelengths, such as 2 or more wavelengths, such as 5 or more different wavelengths, such as 10 or more different wavelengths, such as 25 or more different wavelengths, such as 50 or more different wavelengths, such as 100 or more different wavelengths, such as 200 or more different wavelengths, such as 300 or more different wavelengths, and includes measuring light emitted by a sample in a flowing stream at 400 or more different wavelengths. In some embodiments, two or more detectors in a particle analyzer as described herein are configured to measure collected light at the same or overlapping wavelengths.

[0130] In some embodiments, a fluorescence light detector of interest is configured to measure collected light within a wavelength range (e.g., 200 nm - 1000 nm). In certain embodiments, a detector of interest is configured to collect a spectrum within a wavelength range. For example, a particle analyzer can include one or more detectors configured to collect a spectrum within one or more wavelength ranges of 200 nm–1000 nm. In other embodiments, a detector of interest is configured to measure light emitted by a sample in a flowing stream at one or more specific wavelengths. For example, a particle analyzer can include one or more detectors configured to measure light at one or more of 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785nm, 647 nm, 617 nm, and any combination thereof. In certain embodiments, one or more detectors can be configured to pair with a specific fluorophore, such as a fluorophore used with a sample in a fluorescence assay.

[0131] In some embodiments, the particle analyzer includes one or more wavelength separators positioned between the flow cell and the particle modulated light detector. The term "wavelength separator" is used herein in its conventional sense to refer to an optical component configured to separate light collected from a sample into a predetermined spectral range. In some embodiments, the particle analyzer includes a single wavelength separator. In other embodiments, the particle analyzer includes multiple wavelength separators, such as two or more wavelength separators, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more, such as fifteen or more, such as twenty-five or more, such as fifty or more, such as seventy-five or more, and including one hundred or more wavelength separators. In some embodiments, the wavelength separator is configured to separate light collected from a sample into a predetermined spectral range by passing light having a predetermined spectral range and reflecting light of one or more remaining spectral ranges. In other embodiments, the wavelength separator is configured to separate light collected from a sample into a predetermined spectral range by passing light having a predetermined spectral range and absorbing light of one or more remaining spectral ranges. In other embodiments, the wavelength separator is configured to spatially diffract light collected from a sample into a predetermined spectral range. Each wavelength separator can be any convenient light separation scheme, 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 light detection system is a dichroic mirror.

[0132] Suitable flow cytometry systems can include, but are not limited to, 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. Jan;49(pt 1):17-28; Linden, et al., Semin Throm Hemost. 2004 Oct;30(5):502-11; Alison, et al. J Pathol, 2010 Dec; 222(4):335-344; and Herbig, et al. (2007) Crit Rev Ther Drug Carrier Syst. 24(3):203-255; the disclosures of which are incorporated herein by reference. In some cases, the flow cytometry system of interest includes BD Biosciences FACSCanto TM Flow cytometer, BD Biosciences FACSCanto TM II Flow cytometer, BD Accuri TM Flow cytometer, BD Accuri TM C6 Plus Flow cytometer, BD Biosciences FACSCelesta TM Flow cytometer, BDBiosciences FACSLyric TM Flow cytometer, BD Biosciences FACSVerse TM Flow cytometer, BDBiosciences FACSymphony TM Flow cytometer, BD Biosciences LSRFortessa TM Flow cytometer, BDBiosciences LSRFortessa TM X-20 Flow cytometer, BD Biosciences FACSPresto TM Flow cytometer, BD Biosciences FACSVia TMFlow cytometers and BD Biosciences FACSCalibur TM Cell sorters, BD Biosciences FACSCount TM Cell sorters, BD Biosciences FACSLyric TM Cell sorters, BDBiosciences Via TM Cell sorters, 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 TM S6BD cell sorters, FACSDiscover™ S8 Cell Sorter, etc.

[0133] In some embodiments, the subject system is a flow cytometry system, such as those described in U.S. Patent Nos. 10,663,476, 10,620,111, 10,613,017, 10,605,713, 10,585,031, 10,578,542, 10,578,469, 10,481,074, 10,302,545, 10,145,793, 10,113,967, 10,006,852, 9,952,076, 9,933,341, 9,726,527, 9,453,789, 9,200,334, 9,097,640, 9,095,494, 9,092,034, 8,975,595, 8,753,573, 8,233,146, 8,140,300, 7,544,326, 7,201,875, 7,129,505, 6,821,740, 6,813,017, 6,809,804, 6,372,506, 5,700,692, 5,643,796, 5,627,040, 5,620,842, 5,602,039, 4,987,086, 4,498,766, the disclosures of which are incorporated herein by reference in their entireties.

[0134] In some cases, the flow cytometry system of the present invention is configured to image particles in a flowing stream by fluorescence imaging using fluorescence imaging with radiofrequency tagging (FIRE), such as those described in Diebold, et al., Nature Photonics Vol. 7(10); 806 - 810 (2013) 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, 10,451,538, 10,620,111 and U.S. Patent Publication Nos. 2017 / 0133857, 2017 / 0328826, 2017 / 0350803, 2018 / 0275042, 2019 / 0376895 and 2019 / 0376894, the disclosures of which are incorporated herein by reference. In such cases, the flow cytometry data can include image data of the particles, such as cells present in the sample. See, e.g., Schraivogel et al., Science Vol. 375(6578); 315 - 320 (2022), the disclosure of which is incorporated herein in its entirety, and U.S. Provisional Patent Application Serial No. 63 / 256,974, the disclosure of which is incorporated herein in its entirety. An example of such a system is the FACSDiscover™ S8 Cell Sorter cell sorter.

[0135] In some embodiments, the system is a particle analyzer, wherein the particle analysis system 300 ( Figure 9A ) can be used to analyze and characterize particles, whether or not the particles are physically sorted into a collection container. Figure 9A A functional block diagram of a particle analysis system for computational - based sample analysis and particle characterization is shown. In some embodiments, the particle analysis system 300 is a flow system. Figure 9A The particle analysis system 300 shown therein can be configured to perform all or part of the methods described herein, e.g., The particle analysis system 300 includes a fluid system 302. The fluid system 302 can include or be coupled with a sample tube 310 and a moving fluid column within the sample tube, in which particles 330 (e.g., cells) of the sample move along a common sample path 320.

[0136] The particle analysis system 300 includes a detection system 304 configured to collect signals from each particle as each particle passes through one or more detection stations along a common sample path. The detection station 308 generally refers to the monitored area 340 of the common sample path. In some embodiments, the detection may include detecting light or one or more other characteristics of the particle 330 as it passes through the monitored area 340. In Figure 9A FIG., a detection station 308 with one monitored area 340 is shown. Some embodiments of the particle analysis system 300 may include multiple detection stations. Additionally, some detection stations may monitor multiple regions.

[0137] Each signal is assigned a signal value to form a data point for each particle. As described above, this data may be referred to as event data. The data points may be multi-dimensional data points, including values for the various characteristics measured for the particle. The detection system 304 is configured to collect a series of such data points during a first time interval.

[0138] 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 a 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 in a portion of the first time interval. The control system 306 may additionally compare the experimental signal frequency to the calculated signal frequency or a predetermined signal frequency.

[0139] Figure 9B FIG. shows a system 400 for flow cytometry according to an illustrative embodiment of the present invention. The system 400 includes a flow cytometer 410, a controller / processor 490, and a memory 495. The flow cytometer 410 includes one or more excitation lasers 415a - 415c, a focusing lens 420, a flow cell 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 long pass ("LP") filters 455a - 455b, and one or more fluorescence detectors 460a - 460f.

[0140] The excitation lasers 415a - 415c emit light in the form of laser beams. In Figure 9BIn an example system, the wavelengths of the laser beams emitted by the excitation lasers 415a - 415c are 488 nm, 633 nm, and 325 nm, respectively. First, the laser beams are directed through one or more beam splitters 445a and 445b. Beam splitter 445a transmits light at 488 nm and reflects light at 633 nm. Beam splitter 445b transmits ultraviolet light (light with wavelengths in the range of 10 to 400 nm) and reflects light at 488 nm and 633 nm.

[0141] Then, the laser beams are directed to a focusing lens 420, which focuses the beams onto the portion of the fluid stream within the flow cell 425 where the sample particles are located. The flow cell is part of a fluidics system that guides the particles in the stream (usually one at a time) to the focused laser beam for interrogation. The flow cell can include a flow chamber in a bench - top cytometer or a nozzle head in an air - flow cytometer.

[0142] Light from the laser beams interacts with the particles in the sample through diffraction, refraction, reflection, scattering, and absorption, and is re - emitted at various different wavelengths depending on the characteristics of the particles (such as their size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally present on or within the particles). The fluorescent emission, as well as the diffracted, refracted, reflected, and scattered light, can be routed through one or more beam splitters 445a - 445g, band - pass filters 450a - 450e, long - pass filters 455a - 955b, and a fluorescence collection lens 440 to one or more forward - scatter detectors 430, side - scatter detectors 435, and one or more fluorescence detectors 460a - 460f.

[0143] The fluorescence collection lens 440 collects the light emitted from the particle - laser beam interaction and routes this light to one or more beam splitters and filters. Band - pass filters, such as band - pass filters 450a - 450e, allow a narrow range of wavelengths to pass through the filter. For example, band - pass 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, the 510 / 20 filter extends 10 nm on each side of the spectral - band center, or from 500 nm to 520 nm. Short - pass filters transmit light with wavelengths equal to or shorter than a specified wavelength. Long - pass filters, such as long - pass filters 455a - 455b, transmit light with wavelengths equal to or longer than a specified wavelength. For example, long - pass filter 455a, which is a 670 nm long - pass filter, transmits light with wavelengths equal to or longer than 670 nm. Filters are typically selected to optimize the specificity of the detector for a particular fluorescent dye. The filters can be configured such that the spectral band of the light transmitted to the detector is close to the emission peak of the fluorescent dye.

[0144] The beam splitter directs light of different wavelengths in different directions. The beam splitter can be characterized by filter characteristics such as short pass and long pass. For example, beam splitter 445g is a 620 SP beam splitter, which means that beam splitter 445g transmits light with a wavelength of 620 nm or shorter and reflects light with a wavelength greater than 620 nm in a different direction. In one embodiment, beam splitters 445a - 445g can include optical mirrors, such as dichroic mirrors.

[0145] The forward scatter detector 430 is located slightly off - axis from the direct beam passing through the flow cell and is configured to detect diffracted light, i.e., excitation light that mainly passes forward through or around the particles. The intensity of the light detected by the forward scatter detector depends on the overall size of the particles. The forward scatter detector can include a photodiode. The side scatter detector 435 is configured to detect refracted and reflected light from the particle surface and internal structure and tends to increase as the complexity of the particle structure increases. Fluorescent emission from fluorescent molecules associated with the particles can be detected by one or more fluorescence detectors 460a - 460f. The side scatter detector 435 and the fluorescence detectors can include photomultiplier tubes. Signals detected at the forward scatter detector 430, the side scatter detector 435, and the fluorescence detectors can be converted by the detectors into electrical signals (voltages). This data can provide information about the sample.

[0146] Those skilled in the art will recognize that the flow cytometer according to the embodiments of the present invention is not limited to Figure 9B the flow cytometer shown in, but can include any flow cytometer known in the art. For example, the flow cytometer can have any number of lasers, beam splitters, filters, and detectors with various wavelengths and various different configurations.

[0147] In operation, the operation of the cytometer is controlled by the controller / processor 490, and measurement data from the detectors can be stored in the memory 495 and processed by the controller / processor 490. Although not explicitly shown, the controller / processor 490 is coupled to the detectors to receive output signals from the detectors and can also be coupled to the electrical and electromechanical components of the flow cytometer 400 to control lasers, fluid flow parameters, etc. Input / output (I / O) functionality 497 can also be provided in the system. The memory 495, the controller / processor 490, and the I / O 497 can be provided entirely as integral parts of the flow cytometer 410. In such an embodiment, the display can also form part of the I / O functionality 497 for presenting experimental data to the user of the cytometer 400. Alternatively, some or all of the memory 495, the controller / processor 490, and the I / O functionality can be part of one or more external devices (e.g., a general-purpose computer). In some embodiments, some or all of the memory 495 and the controller / processor 490 can communicate wirelessly or wired with the flow cytometer 410. The controller / processor 490 in combination with the memory 495 and the I / O 497 can be configured to perform various functions related to the preparation and analysis of flow cytometry experiments.

[0148] Figure 9B The system shown in FIG. 4 includes six different detectors that detect fluorescence in six different wavelength bands (which may be referred to herein as the "filter window" of a given detector), which wavelength bands are defined by the configuration of filters and / or beam splitters in the beam path from the flow cell 425 to each detector. Different fluorescent molecules used in flow cytometry experiments will emit light in their own characteristic wavelength bands. The specific fluorescent labels and their associated fluorescence emission bands used for an experiment can be selected to generally coincide with the filter windows of the detectors. However, as more detectors are provided and more labels are used, a perfect correspondence between the filter windows and the fluorescence emission spectra is not possible. Generally, although the peak of the emission spectrum of a specific fluorescent molecule may be within the filter window of a particular detector, some of the emission spectrum of that label will also overlap with the filter windows of one or more other detectors. This can be referred to as spillover. The I / O 497 can be configured to receive data regarding a flow cytometry experiment having a set of fluorescent labels and a plurality of cell populations having a plurality of markers, each cell population having a subset of the plurality of markers. The I / O 497 can also be configured to receive biological data, marker density data, emission spectrum data, data assigning one or more labels to one or more markers, and cytometer configuration data that assign one or more markers to one or more cell populations. Flow cytometry experiment data (e.g., marker spectral characteristics and cytometer configuration data) can also be stored in the memory 495. The controller / processor 490 can be configured to evaluate the assignment of one or more labels to markers.

[0149] Figure 10 FIG. 3 shows a functional block diagram of an example of a particle analyzer control system (e.g., analysis controller 1002) for analyzing and displaying biological events. The analysis controller 1002 can be configured to implement various processes for controlling the graphical display of biological events.

[0150] 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 the 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.

[0151] The analysis controller 1002 can be configured to receive the 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 of a first plot including the biological event data to the display device 1004. The analysis controller 1002 can be further configured to render a region of interest as a gating around a population of biological event data shown by the display device 1004, e.g., overlaid on the first plot. In some embodiments, the gating can be a logical combination of one or more graphical regions of interest drawn on a single parameter histogram or a bivariate plot. In some embodiments, the display can be used to display particle parameters or saturation detector data.

[0152] The analysis controller 1002 can be further configured to display, on the display device 1004, the biological event data within the gating as different from other events in the biological event data outside the gating. For example, the analysis controller 1002 can be configured to render the color of the biological event data contained within the gating as different from the color of the biological event data outside the gating. The display device 1004 can be implemented as a monitor, a tablet computer, a smart phone, or other electronic device configured to present a graphical interface.

[0153] The analysis controller 1002 can be configured to receive a gating selection signal for identifying gating from a first input device. For example, the first input device can be implemented as a mouse 1005. The mouse 1005 can initiate a gating selection signal to the analysis controller 1002 to identify the gating to be displayed on or manipulated through the display device 1004 (e.g., providing a click on or within the desired gating when the cursor is located on the desired gate). In some embodiments, the first device can be implemented as a keyboard 1006 or other device for providing an input signal to the analysis controller 1002, such as a touch screen, a stylus, an optical detector, or a voice recognition system. Some input devices can include multiple input functions. In such embodiments, each input function can be regarded as an input device. For example, as Figure 10 shown, the mouse 1005 can include a right mouse button and a left mouse button, and each button can generate a trigger event. The trigger event can cause the analysis controller 1002 to change the way the data is displayed, which parts of the data are actually displayed on the display device 1004, and / or provide an input for further processing, such as selecting a population of interest for particle sorting.

[0154] In some embodiments, the analysis controller 1002 can be configured to detect when the mouse 1005 initiates a gating selection. The analysis controller 1002 can be further configured to automatically modify the plot visualization to facilitate the gating process. The modification can be based on a specific distribution of the biological event data received by the analysis controller 1002.

[0155] 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 be further configured to allow the analysis controller 1002 to retrieve the biological event data, such as flow cytometry event data.

[0156] The display device 1004 can be configured to receive display data from the analysis controller 1002. The display data can include a plot of the biological event data and the gating outlining parts of the plot. The display device 1004 can be further configured to change the presented information according to the input received from the analysis controller 1002 together with the input from the particle analyzer 1001, the storage device 1003, the keyboard 1006, and / or the mouse 1005.

[0157] In some embodiments, the analysis controller 1002 can generate a user interface to receive example events for sorting. For example, the user interface can include a controller for receiving example events or example images. Example events or images or example gating can be provided before collecting the event data of the sample or based on an initial set of events of a portion of the sample.

[0158] Figure 11B is a schematic diagram of a particle sorter system 200 (e.g., a particle analyzer or sorting system 202) according to an embodiment presented herein. In some embodiments, the particle sorter system 200 is a cell sorter system. As Figure 11A shown, a droplet formation sensor 202 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 201, which can be coupled to, can include, or can be a nozzle 203. Within the fluid conduit 201, a sheath fluid 204 hydrodynamically focuses a sample fluid 206 containing particles 209 into a moving fluid column 208 (e.g., a stream). Within the moving fluid column 208, the particles 209 (e.g., cells) are arranged in a single file to pass through a monitoring region 211 (e.g., where the laser stream intersects) irradiated by an irradiation source 212 (e.g., a laser). The vibration of the droplet formation sensor 202 causes the moving fluid column 208 to break into a plurality of droplets 210, some of which contain particles 209.

[0159] In operation, a detection station 214 (e.g., an event detector) identifies when a particle of interest (or a cell of interest) passes through 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 break point, notified by a timed droplet delay (Δt), a flash charge can be applied to the moving fluid column 208 such that the droplet of interest is charged. The droplet of interest can include one or more particles or cells to be sorted. The charged droplets can then be sorted by activating deflection plates (not shown) to deflect the droplets into a container (e.g., a collection tube or a porous or microwell sample plate), where the wells or microwells can be associated with specific droplets of interest. As Figure 11A shown, the droplets can be collected in a discharge container 238.

[0160] A detection system 216 (e.g., a droplet boundary detector) is used to automatically determine the phase of the droplet drive signal as an interested particle passes through the monitoring area 211. An exemplary droplet boundary detector is described in U.S. Patent No. 7,679,039, which is incorporated herein by reference. The detection system 216 allows the instrument to accurately calculate the position of each detected particle within the droplet. The detection system 216 can be fed an amplitude signal 220 and / or a phase 218 signal, which in turn is fed (through an amplifier 222) to an amplitude control circuit 226 and / or a frequency control circuit 224. The amplitude control circuit 226 and / or the frequency control circuit 224 in turn controls the droplet formation sensor 202. The amplitude control circuit 226 and / or the frequency control circuit 224 can be included in the control system.

[0161] In some embodiments, the sorting electronics (e.g., the detection system 216, the detection station 214, and the processor 240) can be coupled to a memory configured to store detected events and sorting decisions based thereon. The sorting decisions can include event data for the particles. In some embodiments, the detection system 216 and the detection station 214 can be implemented or communicatively coupled as a single detection unit such that event measurements can be collected by one of the detection system 216 or the detection station 214 and provided to the non-collecting element.

[0162] Figure 11A is a schematic diagram of a particle sorter system according to an embodiment presented herein. Figure 11B The particle sorter system 100 shown in includes deflection plates 152 and 154. Charge can be applied through a flow charging wire in the barb. This generates a stream of droplets 110 containing particles 110 for analysis. The particles can be irradiated with one or more light sources (e.g., lasers) to generate light scattering and fluorescence information. The information of the particles is analyzed, for example, by sorting electronics or other detection systems ( Figure 11A not shown in ). The deflection plates 152 and 154 can be independently controlled to attract or repel charged droplets to direct the droplets towards a target collection container (e.g., one of 172, 174, 176, or 178). As Figure 11A shown, the deflection plates 152 and 154 can be controlled to direct the particles along a first path 162 towards container 174 or along a second path 168 towards container 178. If the particle is not of interest (e.g., does not exhibit scattering or irradiation information within a specified sorting range), the deflection plates can allow the particle to continue along the flow path 164. Such uncharged droplets can enter a waste container through, for example, a suction device 170.

[0163] can include sorting electronics to initiate the collection of measurements, receive the fluorescence signal of the particles, and determine how to adjust the deflection plates to sort the particles. Figure 11AExample embodiments of the embodiments shown include BD FACSAria™ series flow cytometers commercially available from Becton, Dickinson and Company (Franklin Lakes, NJ).

[0164] In some embodiments, the particle sorting system of interest is configured to sort particles using a closed particle sorting module, such as those described in U.S. Patent Publication No. 2017 / 0299493, filed Mar. 28, 2017, the disclosure of which is incorporated herein by reference. In certain embodiments, a sorting decision module having multiple sorting decision units is used to sort particles (e.g., cells) of a sample, such as those described in U.S. Patent Publication No. 2020 / 0256781, the disclosure of which is incorporated herein by reference. In some embodiments, the subject system includes a particle sorting module having deflection plates, such as those described in U.S. Patent Publication No. 2017 / 0299493, filed Mar. 28, 2017, the disclosure of which is incorporated herein by reference.

[0165] In certain embodiments, the system is fluorescence imaging using a particle sorter that enables radio frequency tagged emission imaging, as Figure 12AAs shown. The particle sorter 1200 includes an optical radiation assembly 1200a, which includes a light source 1201 (e.g., a 488 nm laser), which generates an output beam 1201a, which is split into beams 1202a and 1202b by a beam splitter 1202. Beam 1202a propagates through an acousto-optic device (e.g., an acousto-optic deflector, AOD) 1203 to produce an output beam 1203a having one or more angularly deflected beams. In some cases, the output beam 1203a generated from the acousto-optic device 1203 includes a local oscillator beam and a plurality of radio frequency comb beams. Beam 1202b propagates through an acousto-optic device (e.g., an acousto-optic deflector, AOD) 1204 to produce an output beam 1204a having one or more angularly deflected beams. In some cases, the output beam 1204a generated from the acousto-optic device 1204 includes a local oscillator beam and a plurality of radio frequency comb beams. The output beams 1203a and 1204a generated from the acousto-optic devices 1203 and 1204, respectively, are combined with a beam splitter 1205 to produce an output beam 1205a, which is transmitted through an optical component 1206 (e.g., an objective lens) to irradiate the particles in the flow cell 1207. In certain embodiments, the acousto-optic device 1203 (AOD) splits a single laser beam into an array of small beams, each having a different optical frequency and angle. A second AOD 1204 adjusts the optical frequency of the reference beam and then overlaps it with the array of small beams at the beam combiner 1205. In certain embodiments, the optical irradiation system having a light source and an acousto-optic 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 / 04049433, the disclosures of which are incorporated herein by reference.

[0166] The output beam 1205a irradiates the sample particles 1208 propagating through the flow cell 1207 (e.g., having a sheath fluid 1209) at the radiation region 1210. As shown in the radiation region 1210, a plurality of beams (e.g., angularly deflected radio frequency shifted beams depicted in the form of points on the radiation region 1210) overlap with a reference local oscillator beam (depicted in the form of hatched lines on the radiation region 1210). Due to their different optical frequencies, the overlapping beams exhibit beat frequency behavior, which results in each small beam carrying a sine modulation at a different frequency f 1-n carrying a sine modulation.

[0167] Light from the radiation sample is transmitted to a light detection system 1200b that includes a plurality of photodetectors. The light detection system 1200b includes a forward scatter light photodetector 1211 for generating a forward scatter image 1211a and a side scatter light photodetector 1212 for generating a side scatter image 1212a. The light detection system 1200b also includes a bright field photodetector 1213 for generating a light loss image 1213a. In some embodiments, the forward scatter detector 1211 and the side scatter detector 1212 are photodiodes (e.g., avalanche photodiodes, APDs). In some cases, the bright field photodetector 1213 is a photomultiplier tube (PMT). Fluorescence from the radiation sample is also detected using fluorescence photodetectors 1214 - 1217. In some cases, the photodetectors 1214 - 1217 are photomultiplier tubes. Light from the radiation sample is directed through a beam splitter 1220 to the side scatter detection channel 1212 and the fluorescence detection channels 1214 - 1217. The light detection system 1200b includes bandpass optical components 1221, 1222, 1223, and 1224 (e.g., dichroic mirrors) for propagating light of a predetermined wavelength to the photodetectors 1214 - 1217. In some cases, the optical component 1221 is a 534 nm / 40 nm bandpass. In some cases, the optical component 1222 is a 586 nm / 42 nm bandpass. In some cases, the optical component 1223 is a 700 nm / 54 nm bandpass. In some cases, the optical component 1224 is a 783 nm / 56 nm bandpass. The first number represents the center of the spectral band. The second number provides the spectral band range. Thus, a 510 / 20 filter extends 10 nm on each side of the spectral band center, or from 500 nm to 520 nm.

[0168] Data signals generated in response to light detected in the scattered light detection channels 1211 and 1212, bright field light detection channels 1213, and fluorescence detection channels 1214 - 1217 are processed in real-time digitally by processors 1250 and 1251. Based on the data signals generated in processors 1250 and 1251, images 1211a - 1217a can be generated in each light detection channel. In response to a sorting signal generated in sorting trigger 1252, sorting of the enabled images is performed. Sorting assembly 1200c includes deflection plates 1231 for deflecting particles into sample container 1232 or waste stream 1233. In some cases, sorting assembly 1200c is configured to sort particles using a closed particle sorting module, such as those described in U.S. Patent Publication No. 2017 / 0299493, filed on March 28, 2017, the disclosure of which is incorporated herein by reference. In certain embodiments, sorting assembly 1200c includes a sorting decision module having a plurality of sorting decision units, such as those described in U.S. Patent Publication No. 2020 / 0256781, the disclosure of which is incorporated herein by reference.

[0169] Figure 12B Depicts image-enabled particle sorting data processing according to certain embodiments. In some cases, the image-enabled particle sorting data processing is a low-latency data processing pipeline. Each photodetector generates pulses with high-frequency modulation 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). Then, real-time sorting and classification electronics classify the particles based on the image features, generating sorting decisions for selectively charging the droplets.

[0170] Computer control system Aspects of the present disclosure further include a computer control system, where the system includes one or more computers for fully or partially automating the subject methods and systems for particle analysis. In some embodiments, the system includes a computer storing a computer program, where when the computer program is loaded onto the computer, the computer program includes instructions for displaying data according to embodiments of the present invention. In some instances, the system is or includes a flow cytometer.

[0171] The system may include a display and an operator input device. The operator input device may be a keyboard, a mouse, etc. The processing module includes a processor that can access a memory on which instructions for performing the steps of the subject method are stored. The processing module may include an operating system, a graphical user interface (GUI) controller, a system memory, a memory storage device, and an input / output controller, a cache, a data backup unit, and many other devices. The processor may be a commercially available processor or one of other existing or upcoming processors. The processor executes the operating system, and the operating system interfaces with the firmware and hardware in a well-known manner and helps the processor coordinate and execute the functions of various computer programs, which can be written in various programming languages (such as Java, Perl, C++, Python, other high-level or low-level languages, and combinations thereof), as known in the art. The operating system typically coordinates and executes the functions of other components of the computer in cooperation with the processor. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, all in accordance with known techniques. In some embodiments, the processor includes analog electronics that provide feedback control (such as negative feedback control).

[0172] The system memory may be various known or future memory storage devices. Examples include any commonly used random access memory (RAM), magnetic media (such as a resident hard disk or tape), optical media (such as a read / write optical disc), flash memory devices, or other memory storage devices. The memory storage device may be various known or future devices, including an optical disc drive, a tape drive, or a floppy disk drive. This type of memory storage device typically reads from and / or writes to a program storage medium (not shown), such as an optical disc. Any of these program storage media, or other program storage media that are currently in use or may be developed in the future, may be considered a computer program product. As will be understood, these program storage media typically store computer software programs and / or data. The computer software programs (also known as computer control logic) are typically stored in the system memory and / or program storage device used with the memory storage device.

[0173] In some embodiments, a computer program product is described that includes a computer-usable medium having control logic (computer software program, including program code) stored therein. When the control logic is executed by a computer processor, the control logic causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware, such as using a hardware state machine. It will be apparent to those skilled in the relevant art how to implement a hardware state machine to perform the functions described herein.

[0174] The memory can be any suitable device in which the processor can store and retrieve data, such as a magnetic, optical, or solid-state storage device (including a disk or optical disc or magnetic tape or RAM, or any other suitable device, whether fixed or portable). The processor can include a general-purpose digital microprocessor that is appropriately programmed from a computer-readable medium carrying the necessary program code. The programming can be provided to the processor remotely via a communication channel or pre-stored in a computer program product (such as a memory or some other portable or fixed computer-readable storage medium) using any of these devices associated with the memory. For example, a disk or optical disc can carry the program and can be read by a disk writer / reader. The system of the present invention also includes a program, for example in the form of a computer program product, for practicing the algorithms of the above-described methods. The program according to the present invention can 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-ROM; electrical storage media, such as RAM and ROM; portable flash drives; and hybrids of these categories, such as magnetic / optical storage media.

[0175] The processor can also access a communication channel to communicate with a user at a remote location. A remote location means that the user does not directly contact the system, but instead transfers input information from an external device (such as a computer connected to a wide area network (“WAN”), telephone network, satellite network, or any other suitable communication channel, including a mobile phone (i.e., a smartphone)) to the input manager.

[0176] In some embodiments, the system according to the present disclosure can be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or transmitter for communicating with a network and / or another device. The communication interface can be configured for wired or wireless communication, including but not limited to radio frequency (RF) communication (such as radio frequency identification (RFID), Zigbee communication protocol, Wi-Fi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth® communication protocol, and cellular communication, such as code division multiple access (CDMA) or global system for mobile communications (GSM)).

[0177] In one embodiment, the communication interface is configured to include one or more communication ports, such as physical ports or interfaces, such as USB ports, USB-C ports, RS-232 ports, or any other suitable electrical connection port, to allow 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)) that are configured for similar complementary data communication.

[0178] In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol to enable the subject system to communicate with other devices such as computer terminals and / or networks, communication-enabled mobile phones, personal digital assistants, or any other communication devices that the user may use in combination.

[0179] In one embodiment, the communication interface is configured to provide a data transfer connection using the Internet Protocol (IP) over a cellular telephone 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.

[0180] In one embodiment, the subject system is configured to communicate wirelessly with a server device through the communication interface, for example, using common standards such as 802.11 or Bluetooth® RF protocol, or the IrDA infrared protocol. The server device can be another portable device such as a smart phone, personal digital assistant (PDA), or laptop 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 an input device such as buttons, a keyboard, a mouse, or a touch screen.

[0181] In some embodiments, the communication interface is configured to communicate data stored in the subject system (e.g., an optional data storage unit) with a network or server device automatically or semi-automatically using one or more of the above communication protocols and / or mechanisms.

[0182] The output controller may include a controller for any of a variety of known display devices for presenting information to a user, whether human or machine, local or remote. If a display device provides visual information, that information can generally be logically and / or physically organized into an array of image elements. A graphical user interface (GUI) controller may include a variety of known or future software programs for providing a graphical input and output interface between the system and the user and for processing user input. The functional elements of a computer may communicate with each other via a system bus. In alternative embodiments, some of these communications may be implemented using a network or other type of remote communication. The output manager may also provide information generated by the processing module to a user at a remote location, for example, via the Internet, telephone, or satellite network, according to known techniques. The presentation of data by the output manager may be implemented according to a variety of known techniques. As some examples, the data may include SQL, HTML, or XML documents, email, or other files or other forms of data. The data may include Internet URL addresses such that the user may retrieve other SQL, HTML, XML, or other documents or data from a remote source. One or more platforms present in the subject system may be any type of known computer platform or a type to be developed in the future, although they generally fall within a class of computers commonly referred to as servers. However, they may also be mainframe computers, workstations, or other types of computers. They may be connected by any known or future type of cable or other communication system, including wireless systems, whether networked or otherwise. They may be co-located or physically separated. A variety of operating systems may be used on any computer platform, which may depend on the type and / or brand 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 so on.

[0183] Figure 13 Depicts a general architecture of an example computing device 1300 in accordance with certain embodiments. Figure 13The general architecture of the computing device 1300 shown includes an arrangement of computer hardware and software components. However, showing all of these generally conventional elements is not required to provide a workable disclosure. As shown, the computing device 1300 includes a processing unit 1310, a network interface 1320, a computer-readable media drive 1330, an input / output device interface 1340, a display 1350, and an input device 1360, all of which can communicate with each other via a communication bus. The network interface 1320 can provide connectivity to one or more networks or computing systems. Thus, the processing unit 1310 can receive information and instructions from other computing systems or services via the network. The processing unit 1310 can also communicate back and forth with the memory 1370 and further provide output information for 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 the non-transitory memory of the analysis system can display flow cytometry event data to the user. The input / output device interface 1340 can also accept input from the optional input device 1360 (e.g., keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, game pad, accelerometer, gyroscope, or other input device).

[0184] The memory 1370 can contain computer program instructions (grouped as modules or components in some embodiments), which the processing unit 1310 executes to implement one or more embodiments. The memory 1370 generally includes RAM, ROM, and / or other persistent, auxiliary, or non-transitory computer-readable media. The memory 1370 can store an operating system 1372, which provides computer program instructions for use by the processing unit 1310 in the general management and operation of the computing device 1300. Data can be stored in the data storage device 1390. The memory 1370 can further include computer program instructions and other information for implementing aspects of the present disclosure.

[0185] Computer-readable storage medium Aspects of the present disclosure further include a non-transitory computer-readable storage medium having instructions for practicing the subject methods. The computer-readable storage medium can be used on one or more computers to fully or partially automate a system for practicing the methods described herein. In certain embodiments, the instructions according to the methods described herein can be encoded on the computer-readable medium in the form of “programming,” where the term “computer-readable medium” as used herein refers to any non-transitory storage medium that participates in providing instructions and data to a computer for execution and processing. In some cases, when the instructions are executed by a computer or processor, the computer or processor will receive display data according to embodiments of the present invention.

[0186] Examples of suitable non-transitory storage media include floppy disks, hard disks, optical disks, magneto-optical disks, CD-ROMs, CD-Rs, magnetic tapes, non-volatile memory cards, ROMs, DVD-ROMs, Blu-ray disks, solid state disks, flash drives, and network attached storage (NAS), whether these devices are internal or external to the computer. A file containing information can be "stored" on a computer-readable medium, where "stored" means recording the information such that the computer can later access and retrieve the information. The computer-implemented methods described herein can be performed using a program, which can be written in one or more of any number of computer programming languages. Such languages include Java, Python, Visual Basic, and C++ as well as many others.

[0187] Kit Aspects of the present disclosure also include kits. The kit can include, for example, a compensation control composition as described herein. In certain embodiments, the control composition of the kit can include positive control particles as described herein. In some embodiments, the control composition of the kit can include negative control particles as described herein. In some cases, the control composition of the kit can include multiple positive control particles and / or multiple negative control particles.

[0188] The kit can also 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 can also include a buffer. For example, the kit can include a buffer such as a sample buffer, a wash buffer, an assay buffer, etc. The kit can 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-biotin related detection reagents, radioactive labels, gold particles, magnetic labels, etc.).

[0189] In certain embodiments, the kit includes the subject compensation control composition and a packaging configured to contain the composition. The packaging can be a sealed packaging, such as a water vapor-proof container, optionally under airtight and / or vacuum sealing. In some embodiments, the packaging can protect its internal contents (e.g., the subject composition) from light irradiation. In these cases, the packaging can protect it from light wavelengths of any range or spectrum, including, for example, visible light, UV rays, light within and / or near the excitation spectrum of any enclosed dye, etc. In certain cases, the packaging is a sterile packaging, configured to maintain the composition enclosed in the packaging 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 further included buffer or reagent (e.g., amine-reactive dye and / or DNA-binding dye) are included in the same packaging. In other cases, the composition and any further included buffer or reagent are included in separate packagings.

[0190] In addition to the above components, the subject kit can further include instructions for practicing the subject method. In some embodiments, the instructions are for using the compensation control composition of the kit to determine the compensation value for cell viability data obtained from flow cytometry analysis. In some embodiments, the instructions are for performing flow cytometry analysis and compensating the obtained cell viability data using the determined compensation value. These instructions can exist in various forms in the subject kit, and one or more of them can be present in the kit. One of the forms in which these instructions can exist is as printed information on a suitable medium or substrate (e.g., one or more sheets of paper printed with the information), the packaging of the kit, a packaging insert, etc. Another means would be a computer-readable medium (e.g., as described above) on which the information can be recorded or stored. Yet another form that can exist is a website address, through which the information at the removed site can be accessed via the Internet. Any convenient form of instructions can be present in the kit.

[0191] Use The subject composition and method can be used in applications that require cell analysis of biological samples for research, laboratory testing, or for therapy. In some embodiments, the subject system and method facilitate the analysis of cells obtained from fluid or tissue samples (e.g., samples of diseases, including but not limited to cancer). The compositions and methods of the present disclosure also allow for the analysis of cells from biological samples (e.g., organs, tissues, tissue fragments, fluids) with higher efficiency and at lower cost.

[0192] The subject control compositions and methods can be used in applications that require the analysis of samples using ultraviolet (UV) or violet lasers. Embodiments of the subject control compositions and methods can be used in applications that require the analysis of samples using two or more fluorescent particles (e.g., two or more dye compositions). For example, the subject compositions and methods can be used in applications that require assays using amine-reactive dyes and DNA-binding dyes (e.g., cell viability assays). In some cases, the subject compositions and methods allow for fluorescence compensation for assays that include one or more of: amine-reactive dyes, DNA-binding dyes, fluorescently labeled nucleic acid probes, and fluorescently labeled antibodies.

[0193] The subject control compositions and methods can be used in situations that require flow cytometry assays with more precise and / or accurate data. For example, compared to prior art protocols and control beads with positive compensation controls having lower fluorescence intensity or negative control samples having higher fluorescence intensity, the subject compositions and methods can facilitate accurate data collection and interpretation. Additionally, compared to prior art protocols and control beads that cannot include a fluorescence minus one (FMO) control for an assay that includes two or more of: amine-reactive dyes, DNA-binding dyes, fluorescently labeled nucleic acid probes, and fluorescently labeled antibodies, the subject control compositions and methods can facilitate accurate data collection and interpretation.

[0194] The subject compositions and methods can be used in laboratory and research workflows that require a high degree of flexibility. For example, the subject compositions and methods can facilitate a laboratory's ability to perform customized experiments on a variety of different cell types. Additionally, compared to prior art control beads with poor stability, the subject compositions and methods can facilitate cost reduction. Embodiments of the subject compositions and methods allow for fluorescence compensation without depleting any cell samples for use as controls. Thus, the subject compositions and methods can also be used in situations that require the analysis of as many collected cell samples as possible (e.g., due to low sample availability, to save costs, or due to possible difficulties in sample collection).

[0195] Examples From the disclosure provided above, it can be seen that the embodiments of the present disclosure have a wide range of applications. Accordingly, the following examples are presented to provide a complete disclosure and description to those of ordinary skill in the art of how to make and use the 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 conducted. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise noted, temperatures are in degrees Celsius and pressures are near atmospheric. Additionally, common laboratory protocol abbreviations can be used (e.g., µl = microliter, min = minute, kcal = kilocalorie, mol = mole, etc.).

[0196] The following experiments demonstrated the stability of the cell viability compensation (CVC) beads of the present invention and compared the positive and negative CVC beads with prior art compensation beads.

[0197] Materials and Methods According to an embodiment of the present invention, an embodiment of a compensation control composition having both positive control beads and negative control beads was produced. Positive control beads were synthesized according to an embodiment of the present invention and comprise: a silica core, a 5-layer histone coating layer, and a 5-layer dsDNA molecule coating layer. For the positive control, the dsDNA coating layer was first covalently attached to the core. Then the histone layer of the coating layer and the dsDNA layer of the coating layer were alternately covalently attached to the outermost coating layer of the positive control beads until there were 5 layers of each. Negative control beads were synthesized according to an embodiment of the present invention and comprise a silica core but lack an aminated polymer (i.e., histone) coating layer and a nucleic acid (i.e., dsDNA) coating layer.

[0198] Figure 3 A method for synthesizing positive control beads was described. Aminated silica cores were obtained and their multiple amine groups were thiolated. Then a dsDNA molecule coating layer modified to contain maleimide groups and a histone coating layer modified to contain thiol groups were alternately deposited onto the positive control beads until there were 5 layers of each.

[0199] Example 1: Staining with amine-reactive dyes Experiments were conducted to compare the fluorescence generated by staining with amine-reactive dyes between an embodiment of the CVC beads of the present invention (see "Materials and Methods" above) and two types of beads of the prior art (ViaComp® (Slingshot) beads and Arc™ (Invitrogen) beads). ViaComp® (Slingshot) beads consist of hydrogels, while Arc™ (Invitrogen) beads are specially modified polystyrene microspheres. Staining was performed using the following three different BD Horizon™ Fixable Viability Staining (FVS) amine-reactive dyes: 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) and 1 µl of FVS dye were incubated in DMSO buffer for 30 min. Data were collected with the same flow cytometer parameter settings.

[0200] Figures 4 - 6 The data shown in indicate that compared with the prior art ViaComp® and Arc™ beads, the CVC beads of the present invention exhibit brighter or comparable staining signals. However, preliminary experiments indicate that the fluorescence intensity signal of the stained CVC beads of the present invention can be tuned or adjusted (e.g., increased) by controlling the number of layers of the histone coating. In addition, unlike the CVC beads, the polystyrene of the Arc™ beads exhibits significant autofluorescence, resulting in different spectral characteristics of the beads after being stained with the cell viability dye compared to the stained cell samples. This problem is particularly evident for the fluorescence emission of the purple and ultraviolet lasers. This significant autofluorescence can lead to misleading spillover values of the dye (especially dyes that emit or absorb light in the UV and purple ranges), which may compromise the compensation data of the experiment.

[0201] The CVC beads of the present invention also exhibit logical advantages over the Arc™ beads. Different from the embodiments of the CVC beads, the Arc™ beads are divided into two vials: one vial of positive beads and one vial of negative control beads. This division increases the complexity and production cost, and further brings inconvenience to the user during the experiment. In addition, the Arc™ beads can only be stained with amine-reactive dyes because they are not reactive with DNA intercalating dyes. This limits the options for the user when designing experiments.

[0202] Example 2: Staining with DNA-binding dyes Experiments were conducted to compare the fluorescence generated by staining with DNA intercalating dyes between the embodiments of the CVC beads of the present invention (see "Materials and Methods" above) and the prior art ViaComp® (Slingshot) hydrogel beads. The following 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)(Fluorescent channel: BUV450). For staining, 50 µl of bead suspension (i.e., both positive and negative beads) and 20 µl of either BDPharmingen™ 7-AAD or 1 µl of BD Pharmingen™ DAPI were incubated in solution for 10 min. Data were collected with the same flow cytometer parameter settings.

[0203] Figure 7 The data shown in indicate that the CVC beads of the present invention exhibit brighter or similar staining signals compared to the prior art ViaComp® beads. However, preliminary experiments indicate that the fluorescence intensity signal of the stained CVC beads of the present invention can be tuned or adjusted (e.g., increased) by controlling the number of layers of the dsDNA coating. In addition, unlike the CVC beads, we observed a higher non-specific uptake of the DNA intercalating dye on the ViaComp® negative control beads. This non-specific uptake, likely due to the hydrogel composition of the beads, can result in high background noise - thus potentially reducing the accuracy of flow cytometry results obtained using the ViaComp® beads.

[0204] Example 3: Control Composition Stability Experiments were conducted to test the stability of the embodiments of the CVC beads of the present invention (see "Materials and Methods" above). The CVC beads were stored at 37°C for 15 days, with subsets of the beads being tested on days 1 - 3, days 7 - 10, day 13, and day 15. For testing, the beads were stained with BD Pharmingen™ 7-AAD, BD Horizon™ FVS570, or BD Horizon™ FVS700, and the fluorescence intensity of the beads stained with each dye was measured. The MFI of the beads stained with each dye on each test day can be seen in Figure 8 .

[0205] Figure 8 The data shown in indicate that the CVC beads of the present invention can be stored at relatively high temperatures for several weeks without a significant reduction in dye staining performance. Using the Arrhenius equation (Q 10 = 3, Ea = 19.4 kcal / mol), it is predicted that the CVC beads can still be effectively stained with amine-reactive dyes and DNA intercalating dyes after storage at 4°C for 18 months. Thus, the CVC beads have a significant advantage over prior art beads (such as ViaComp® beads), which have a shelf life of only 6 months at 2 - 8°C and thus must be transported and stored at -20°C. This temperature instability is inconvenient for users as the beads must be thawed before use and expired beads must be replaced - thus resulting in additional costs.

[0206] Notwithstanding the appended claims, the present disclosure is also defined by the following articles: 1. A positive control particle, comprising: A core; An aminated polymer comprising amines configured to react with amine-reactive dyes; 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.

[0207] 2. The positive control particle according to article 1, wherein the core is substantially non-fluorescent.

[0208] 3. The positive control particle according to article 1 or 2, wherein the core has low non-specific binding.

[0209] 4. The positive control particle according to any one of the preceding articles, wherein the core comprises an inorganic material.

[0210] 5. The positive control particle according to article 4, wherein the core comprises silica.

[0211] 6. The positive control particle according to any one of articles 1 to 3, wherein the core comprises a polymer.

[0212] 7. The positive control particle according to article 6, wherein the core comprises poly(methyl methacrylate) (PMMA).

[0213] 8. The positive control particle according to article 6, wherein the core comprises a polyacrylamide hydrogel.

[0214] 9. The positive control particle according to any one of the preceding articles, wherein the diameter of the particle ranges from 0.1 to 150 microns.

[0215] 10. The positive control particle according to article 9, wherein the diameter of the particle ranges from 1 to 10 microns.

[0216] 11. The positive control particle according to article 10, wherein the diameter of the particle ranges from 5 to 7 microns.

[0217] 12. The positive control particle according to any one of the preceding articles, wherein the aminated polymer and the nucleic acid are laminated on the core.

[0218] 13. The positive control particle according to article 12, wherein the particle comprises 5 or more aminated polymer layers and 5 or more nucleic acid layers.

[0219] 14. The positive control particles according to any one of the preceding items, wherein the aminated polymer comprises a polypeptide.

[0220] 15. The positive control particles according to item 14, wherein the polypeptide is a protein.

[0221] 16. The positive control particles according to item 15, wherein the protein is a histone.

[0222] 17. The positive control particles according to item 15, wherein the polypeptide is myelin basic protein.

[0223] 18. The positive control particles according to any one of items 1 to 14, wherein the aminated polymer comprises a polysaccharide.

[0224] 19. The positive control particles according to item 18, wherein the polysaccharide is a cationic polymer.

[0225] 20. The positive control particles according to item 19, wherein the cationic polymer is chitosan.

[0226] 21. The positive control particles according to any one of the preceding items, wherein the nucleic acid is a double-stranded nucleic acid.

[0227] 22. The positive control particles according to any one of the preceding items, wherein the nucleic acid is deoxyribonucleic acid (DNA).

[0228] 23. The positive control particles according to any one of the preceding items, wherein the size of the nucleic acid ranges from 450 to 550 base pairs.

[0229] 24. The positive control particles according to any one of the preceding items, wherein the nucleic acid is naturally occurring.

[0230] 25. The positive control particles according to item 24, wherein the nucleic acid comprises salmon DNA.

[0231] 26. The positive control particles according to any one of items 1 to 23, wherein the nucleic acid is synthetic.

[0232] 27. The positive control particles according to any one of the preceding items, wherein the aminated polymer is covalently bound to the surface of the core directly or via a linker with the nucleic acid.

[0233] 28. The positive control particles according to any one of the preceding items, wherein the particles are beads.

[0234] 29. A compensation control composition comprising a plurality of positive control particles according to any one of the preceding items.

[0235] 30. The compensated control composition according to item 29, further comprising a plurality of negative control particles.

[0236] 31. The compensated control composition according to item 30, wherein the negative control particles comprise a core, and the core comprises the same material and the same average diameter as the core of the positive control particles.

[0237] 32. The compensated control composition according to item 30 or 31, wherein polyethylene glycol (PEG) is conjugated to the core of the negative control particles.

[0238] 33. The compensated control composition according to any one of items 29 to 32, wherein the composition is stable at 4 degrees Celsius for 18 or more months.

[0239] 34. The compensated control composition according to item 1, further comprising the amine-reactive dye.

[0240] 35. A labeled compensated control composition, comprising a plurality of positive control particles according to any one of items 1 to 28, wherein the positive control particles have an amine-reactive dye and / or a DNA-binding dye bound thereto.

[0241] 36. The labeled compensated control composition according to item 35, wherein the amine-reactive dye is a cell viability dye.

[0242] 37. The labeled compensated control composition according to item 35 or 36, wherein the maximum excitation wavelength range of the amine-reactive dye is from 350 nm to 1000 nm.

[0243] 38. The labeled compensated control composition according to any one of items 35 to 37, wherein the amine-reactive dye is selected from BD Horizon™ Fixable Viability Stains, Biolegend Zombie™ Dyes, Thermo FischereFluor™ Dyes, Thermo Fischer LIVE / DEAD™ Stains, Proteintech Phantom Dyes, and TonboBiosceinces Ghost Dye™.

[0244] 39. The labeled compensated control composition according to any one of items 35 to 38, wherein the DNA-binding dye is a cell viability dye.

[0245] 40. A labeled compensation control composition according to any one of items 35 to 39, wherein the maximum excitation wavelength range of the DNA-binding dye is from 350 nm to 1000 nm.

[0246] 41. A labeled compensation control composition according to any one of items 35 to 40, wherein the DNA-binding dye is selected from 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.

[0247] 42. A labeled compensation control composition according to any one of items 35 to 41, further comprising a plurality of negative control particles.

[0248] 43. The labeled compensation control composition according to item 42, wherein the negative control particles comprise a core, and the core comprises the same material and the same average diameter as the core of the positive control particles.

[0249] 44. The labeled compensation control composition according to item 43, wherein PEG is conjugated to the core of the negative control particles.

[0250] 45. A method for determining a compensation value for cell viability data obtained from flow cytometry analysis, the method comprising: Analyzing a labeled compensation control composition according to any one of items 35 to 44 using a flow cytometer to obtain flow cytometry data; and Calculating a compensation value based on the flow cytometry data.

[0251] 46. The method according to item 45, further comprising preparing the labeled compensation control composition.

[0252] 47. The method according to item 46, wherein preparing comprises contacting a compensation control composition according to any one of items 29 to 34 with an amine-reactive dye and / or a DNA-binding dye.

[0253] 48. The method according to any one of items 41 to 47, further comprising compensating the cell viability data, the compensation comprising: Contacting a cell sample with the same dye conjugated to the positive control particles, Generating the cell viability data by analyzing the cell sample using a flow cytometer; and Modifying the cell viability data using the calculated compensation values.

[0254] 49. A method of generating positive control particles, the method comprising: Covalently associating an aminated polymer and a nucleic acid with a core to produce positive control particles.

[0255] 50. The method according to item 49, wherein the aminated polymer and the nucleic acid are layered on the surface of the core.

[0256] 51. A kit, comprising: The compensation control composition according to any one of items 29 to 34.

[0257] 52. The kit according to item 51, wherein the kit further comprises an amine-reactive dye.

[0258] 53. The kit according to any one of items 51 or 52, wherein the kit further comprises a DNA-binding dye.

[0259] In at least some of the previously described embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment, unless such substitution is technically infeasible. Those skilled in the art should understand that various other omissions, additions, and modifications may be made to the above methods and structures without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter defined by the appended claims.

[0260] Those skilled in the art should understand that, generally speaking, the terms used herein, especially the terms used in the appended claims (e.g., the subject matter of the appended claims), are usually intended to be "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 "containing" should be interpreted as "containing but not limited to", etc.). Those skilled in the art should also understand that if the intention is to introduce a specific number of recited claims, then such intention will be explicitly recited in the claims, and in the absence of such recitation, there is no such intention. For example, for the sake of understanding, the following appended claims may contain the use of introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed as implying that introducing a claim recitation by the indefinite article "a" or "an" will limit any particular claim containing such introduced claim recitation to only those embodiments containing only one such recitation, even when the same claim includes an introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted as meaning "at least one" or "one or more"); this also applies to the case of using a definite article to introduce a claim recitation. Additionally, even if the specific number of the introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted as meaning at least the recited number (e.g., the mere recitation "two recited things" without other modifiers means at least two recited things, or two or more recited things). Furthermore, in those cases where a convention similar to "at least one of A, B, and C, etc." is used, generally speaking, such construction is intended to be understood by those skilled in the art as to the meaning of the convention (e.g., "a system having at least one of A, B, and C" will include but not be limited to the following systems: A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Moreover, in those cases where a convention similar to "at least one of A, B, or C, etc." is used, generally speaking, such construction is intended to be understood by those skilled in the art as to the meaning of the convention (e.g., "a system having at least one of A, B, or C" will include but not be limited to the following systems: A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Those skilled in the art should also understand that in fact any disjunctive word and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood as contemplating the possibility of including one of the terms, any one of the terms, or both terms. For example, the phrase "A or B" will be understood as including the possibility of "A" or "B" or "A and B".

[0261] In addition, where features or aspects of the present disclosure are described in terms of a Markush group, those skilled in the art will recognize that the present disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0262] As will be understood by those skilled in the art, for any and all purposes, such as to provide a written description, all ranges disclosed herein also cover any and all possible subranges and combinations of subranges thereof. Any listed range can be readily regarded as sufficiently described and enables the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. By way of non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third, and upper third, etc. As will also be understood by those skilled in the art, all such language as “up to,” “at least,” “greater than,” “less than,” and the like include the recited number and refer to ranges that can then be broken down into subranges as described above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1 - 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 - 5 items refers to a group having 1, 2, 3, 4, or 5 items, and so on.

[0263] Although the foregoing invention has been described in some detail for purposes of clarity of understanding by way of illustration and example, it will be apparent to those of ordinary skill in the art that certain changes and modifications may be made without departing from the spirit or scope of the appended claims.

[0264] Accordingly, the foregoing merely illustrates the principles of the invention. It is to be understood that those skilled in the art will be able to devise various arrangements, which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Moreover, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventor to further the art and are to be construed as not being limited to such specifically recited examples and conditions. Further, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof are intended to cover both structural and functional equivalents thereof. Additionally, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed regardless of structure that perform the same function. Moreover, nothing disclosed herein is intended to be dedicated to the public, whether or not such disclosure is expressly recited in the claims.

[0265] Accordingly, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention are embodied by the appended claims. In the claims, 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is expressly defined as being invoked for such a limitation in a claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of a recited limitation in the claim; if such exact phrase is not used in a limitation of the claim, then 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is not invoked.

Claims

1. A positive control particle, comprising: A core; An aminated polymer, the aminated polymer comprising an amine configured to react with an amine-reactive dye; and A nucleic acid, the nucleic acid being configured to bind a DNA-binding dye; Among them, The aminated polymer and the nucleic acid are covalently associated with the core.

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

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

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

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

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

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

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

9. The positive control particle according to any one of the preceding claims, wherein the size of the nucleic acid ranges from 450 to 550 base pairs.

10. The positive control particle according to any one of the preceding claims, wherein the aminated polymer covalently binds to the surface of the core directly or through a linker with the nucleic acid.

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

12. A compensation control composition, comprising a plurality of positive control particles according to any one of the preceding claims.

13. The compensation control composition according to claim 12, further comprising a plurality of negative control particles.

14. A labeled 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. A method for determining a compensation value for cell viability data obtained from flow cytometry analysis, the method comprising: Analyzing the labeled compensation control composition according to claim 14 using a flow cytometer to obtain flow cytometry data; And Calculating a compensation value based on the flow cytometry data.

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