Compositions and methods for cell-like calibration particles

Abstract

The method includes calibrating a cytometry device for analysis of target cells by inserting hydrogel particles into the cytometry device. The hydrogel particles have at least one background fluorescence characteristic or spectral characteristic substantially similar to at least one background fluorescence characteristic or spectral characteristic of the target cells. The method also includes measuring at least one characteristic of the hydrogel particles using the cytometry device.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 965,494, filed January 24, 2020, entitled "Compositions and Methods for Cell-Like Calibration Particles," the entire disclosure of which is incorporated herein by reference.

[0002] This application is related to U.S. Patent No. 9,915,598, issued March 13, 2018, entitled "Hydrogel Particles with Tunable Optical Properties," and U.S. Patent No. 9,714,897, issued July 25, 2017, entitled "Hydrogel Particles with Tunable Optical Properties and Methods for Using the Same," the entire disclosures of each of which are incorporated herein by reference for all purposes.

[0003] Field The present disclosure relates to flow cytometry, and more particularly to hydrogel bead substrates that exhibit cell-like autofluorescence, allowing for more accurate fluorescence and spectral calibration and correction. [Background technology]

[0004] background Flow cytometry and hematological analysis are techniques that enable the rapid separation, enumeration, and characterization of individual cells and are routinely used in clinical and laboratory settings for a variety of applications. This technology is based on directing a beam of light at a focused liquid stream. In some configurations, multiple detectors are then directed at the point where the stream passes through the light beam: one detector aligned with the light beam (forward scatter, or "FSC") and several detectors perpendicular to the light beam (side scatter, or "SSC"). FSC generally correlates with cell volume, while SSC depends on the particle's internal complexity or granularity (i.e., nuclear shape, amount and type of cytoplasmic granules, or membrane roughness). As a result of these correlations, different specific cell types exhibit different FSC and SSC, allowing cell types to be distinguished in flow cytometry. These measurements form the basis of cytometric analysis. In other forms of cytometry, cells are imaged and descriptive characteristics of the cells, such as size, shape, volume, and possibly biochemical characteristics, are recorded. In addition to these measurements, cells are often analyzed with multiple fluorescence channels or spectral analyzers. These detection modes are used to identify biomarker profiles and other biological characteristics among different cell populations.

[0005] Most synthetic or polymeric products used in cellular analysis are made from plastic materials such as polystyrene (latex), an opaque polymer with fixed forward and side scattering profiles generally based on particle diameter. Furthermore, polystyrene has high autofluorescence in important detection channels, even in the absence of fluorophores or associated biomarkers, resulting in background fluorescence signals. In other cases, polystyrene has much lower autofluorescence than cellular materials, leading to inaccurate correction and spectral unmixing. Overall, the inherent autofluorescence of polystyrene often makes it unsuitable for fluorescence calibration and correction. Specifically, rare or low-expressing biomarkers cannot be properly distinguished from polystyrene particles, preventing their use as controls / standards. Furthermore, autofluorescence from polystyrene particles can result in spurious fluorescence resonance energy transfer (FRET), which contributes to insufficient signal-to-noise in dyes that rely on FRET for functionality (e.g., tandem dyes). In spectral analysis, the autofluorescence interference caused by polystyrene is exacerbated, distorting the complete spectral profile of a given target relative to the separated fluorescence channels. Together, these inherent limitations of polystyrene make it a suboptimal substrate for calibration and correction with a range of fluorescent dyes, particularly those that exhibit excitation or emission profiles in the violet and ultraviolet ranges.

[0006] Several key cytometry instrument setup procedures are based on the ability of calibration particles to mimic cells as closely as possible. In cytometry, compensation is the mathematical correction of signal overlap between channels of different fluorescent dyes' emission spectra. When assaying diverse biochemical targets using multiple unique fluorophores, compensation is important because it is crucial to distinguish true signal response from "spillover" signals or interference from different fluorescent channels. In some known configurations, fluorescence compensation uses polystyrene-based controls to demonstrate the fluorescence resolution of a given panel of antibodies / fluorophores. However, due to the autofluorescence of polystyrene, there are entire classes of fluorophores (e.g., tandem dyes, UV / violet-responsive dyes), many of which cannot be effectively compensated for with existing bead-based polystyrene products. Polystyrene autofluorescence and poor performance fundamentally limit the complexity and diversity of fluorophores used during cellular analysis.

[0007] Therefore, there is a need for substrates that more closely mimic the autofluorescence of real cells. Summary of the Invention [Means for solving the problem]

[0008] overview In some embodiments, the method includes calibrating a cytometry device for analysis of target cells by inserting hydrogel particles into the cytometry device. The hydrogel particles have at least one of an autofluorescence characteristic and a spectral characteristic substantially similar to at least one of the autofluorescence characteristic and the spectral characteristic of the target cells. The method also includes measuring at least one characteristic of the hydrogel particles using the cytometry device.

[0009] In some embodiments of the present disclosure, the composition comprises hydrogel particles that have an autofluorescence profile or spectral profile that is more similar to that of cells compared to the autofluorescence profile or spectral profile of polystyrene (e.g., latex) as measured by a cytometry device.

[0010] In other embodiments, the present disclosure provides methods for producing hydrogel particles having autofluorescence or spectral properties substantially similar to the corresponding autofluorescence or spectral properties of target cells. The present disclosure also describes methods for producing hydrogel particles having predetermined autofluorescence and / or spectral properties. The present disclosure also describes methods for calibrating a cytometry device for analysis of target cells, the method comprising: a) inserting into the cytometry device hydrogel particles having autofluorescence and / or spectral properties substantially similar to the corresponding autofluorescence and / or spectral properties of target cells; and b) using the cytometry device to measure the fluorescence and / or spectral properties of the hydrogel particles, thereby calibrating the cytometry device for analysis of the target cells.

[0011] In some embodiments, a method includes calculating a correction value for cytometric measurements of target cells and correcting the cytometric measurements of the target cells based on the correction value. Calculating the correction value for the cytometric measurements of the target cells includes inserting first hydrogel particles into a cytometry device at a first time. The first hydrogel particles have at least one background fluorescence characteristic or spectral characteristic substantially similar to at least one background fluorescence characteristic or spectral characteristic of the target cells. The at least one characteristic of the first hydrogel particles is measured using the cytometry device. The calculating also includes inserting second hydrogel particles into the cytometry device at a second time, different from the first time, and measuring at least one characteristic of the second hydrogel particles using the cytometry device. The calculating also includes comparing the measured at least one characteristic of the first hydrogel particles with the measured at least one characteristic of the second hydrogel particles to determine the correction value. In an embodiment of the present invention, for example, the following items are provided: (Item 1) 1. Calibrating a cytometry device for analysis of target cells, comprising: inserting into the cytometry device hydrogel particles having at least one background fluorescent characteristic or spectral characteristic substantially similar to at least one background fluorescent characteristic or spectral characteristic of the target cells; measuring at least one characteristic of the hydrogel particles using the cytometry device; Calibrate by A method comprising: (Item 2) Prior to inserting the hydrogel particles into the cytometry device, binding a fluorophore-containing reagent to the hydrogel particles to form a complex; measuring at least one property of the complex; and calculating fluorescence correction or spectral unmixing based on said at least one measured property; and Item 1, the method of claim 1 further comprising: (Item 3) 2. The method of claim 1, wherein the hydrogel particles are modified to bind to an antibody that binds a conjugated fluorophore. (Item 4) 4. The method of claim 3, wherein the conjugated fluorophore is a fluorescent dye. (Item 5) 2. The method of claim 1, wherein the hydrogel particles are modified hydrogel particles that have been modified to bind to at least one of an intercalating nucleic acid labeling reagent or an amine-reactive nucleic acid labeling reagent. (Item 6) 6. The method of claim 5, further comprising assessing the viability of the target cells using the modified hydrogel particles. (Item 7) Item 10. The method of claim 1, wherein the hydrogel particles have a refractive index greater than about 1.15. (Item 8) Item 10. The method of item 1, wherein the hydrogel particles have a refractive index greater than about 1.3. (Item 9) Item 10. The method of item 1, wherein the hydrogel particles have a refractive index greater than about 1.7. (Item 10) Item 10. The method of item 1, wherein the hydrogel particles have a diameter of less than about 100 μm. (Item 11) Item 1, wherein the hydrogel particles have a diameter of less than about 10 μm. (Item 12) Item 10. The method of item 1, wherein the hydrogel particles have a diameter of less than about 1 μm. (Item 13) Item 10. The method of item 1, wherein the hydrogel particles contain a polymeric nanoparticle additive. (Item 14) Item 10. The method of claim 1, wherein the hydrogel particles are chemically functionalized hydrogel particles. (Item 15) 2. The method of claim 1, wherein the hydrogel particles comprise free amine groups. (Item 16) Item 10. The method of claim 1, wherein the hydrogel particles comprise allylamine. (Item 17) 2. The method of claim 1, wherein the target cell is an immune cell. (Item 18) Item 10. The method of claim 1, further comprising polymerizing the droplets to produce the hydrogel particles. (Item 19) Item 10. The method of claim 1, wherein the hydrogel particles are modified by conjugating or attaching one of a fluorophore or a fluorescent dye. (Item 20) 20. The method of claim 19, wherein the modified hydrogel particles match the fluorescence or spectral profile of a cell. (Item 21) Item 10. The method of item 1, wherein the at least one characteristic comprises one of an inter-laser delay, a fluorescence response, a sort timing, or a fluorescence compensation. (Item 22) Calculating a plurality of adjustment values ​​for cytometric measurements of target cells, the adjustment values ​​comprising: inserting into a cytometry device first hydrogel particles and second hydrogel particles, the first hydrogel particles having at least one background fluorescent characteristic or spectral characteristic substantially similar to at least one background fluorescent characteristic or spectral characteristic of the target cells, and the second hydrogel particles either configured to bind to or pre-bound to a reagent, the reagent being a reagent that generates at least one fluorescent signal that is different from the background fluorescent characteristic or the spectral characteristic; measuring at least one property of the first hydrogel particles and at least one property of the second hydrogel particles using the cytometry device; comparing the measured at least one property of the first hydrogel particles with the measured at least one property of the second hydrogel particles to determine a fluorescence overlap with at least one additional reagent and a spectral overlap with the at least one additional reagent; Calculating by, and modifying the cytometric measurement of the target cells based on the plurality of adjustment values; A method comprising: [Brief explanation of the drawings]

[0012] [Figure 1-1] FIG. 1A shows exemplary optical properties of (A) hydrogel particles of the present disclosure, (B) polystyrene beads, according to some embodiments.

[0013] [Figure 1-2] FIG. 1B shows the optical properties of polystyrene beads in contrast to the optical properties of the hydrogel particles in FIG. 1A(A).

[0014] [Figure 2] FIG. 2 shows the difference in scattering profiles between polystyrene and real / target cells or hydrogel particles of the present disclosure, according to some embodiments.

[0015] [Figure 3] FIG. 3 illustrates how the autofluorescence or spectral properties of a hydrogel, according to some embodiments, can be engineered to match the autofluorescence or spectral properties of a target cell population.

[0016] [Figure 4] FIG. 4 illustrates the ability to independently tune each of the engineered hydrogel properties to match the passive light scattering, autofluorescence, biomarker, and fluorescence properties of any target cell, according to some embodiments.

[0017] [Figure 5] FIG. 5 illustrates an exemplary method for producing hydrogel particles, according to some embodiments.

[0018] [Figure 6] FIG. 6 is a plot of intensity versus wavelength / channel demonstrating the principle of fluorescence compensation.

[0019] [Figure 7-1]7A-7C include plots of data showing a comparison of human cells to polystyrene particles and hydrogel particles across a range of fluorescence and spectral detectors, according to some embodiments. [Figure 7-2] Same as above. [Figure 7-3] Same as above.

[0020] [Figure 8-1] FIG. 8A is a spectral profile of a lymphocyte stained with an Alexa 700-modified antibody (“Ab”) according to some embodiments, and FIG. 8B is a spectral profile of a hydrogel particle of the present disclosure according to some embodiments, showing their alignment with the spectral profile of the lymphocyte in FIG. 8A. [Figure 8-2] Same as above.

[0021] [Figure 9] FIG. 9 is a flow diagram illustrating a method for calibrating a cytometry device for analysis of target cells, according to some embodiments.

[0022] [Figure 10] FIG. 10 is a flow diagram illustrating a process for calibration and calculation of fluorescence compensation and / or spectral unmixing using hydrogels of the present disclosure, according to some embodiments.

[0023] [Figure 11-1] 11A-11D are bar graphs showing a comparison between cell staining, according to some embodiments, hydrogel compensation beads, and known (polystyrene-based) products. [Figure 11-2] Same as above. [Figure 11-3] Same as above. [Figure 11-4] Same as above.

[0024] [Figure 12]FIG. 12 is a chart comparing the autofluorescence of polystyrene and cell controls with the autofluorescence of exemplary hydrogels of the present disclosure having various compositions and properties, according to some embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0025] Detailed Description Several known calibration measurements for flow cytometers, such as inter-laser delay, fluorescence response, sort timing, and fluorescence compensation, use polystyrene beads. These calibration measurements can be crucial for the accurate operation of the cytometer and for any downstream analysis or sorting of cell populations. While polystyrene is robust and low-cost compared to the use of cell controls, it exhibits inherently different optical and fluorescent behavior compared to cells. As a result, polystyrene beads are a poor substitute for cell controls in all but the most basic calibration processes.

[0026] To overcome the limitations of polystyrene, cells are sometimes used during instrument setup and calibration, but such approaches suffer from batch-to-batch variability, high cost, short shelf life, and biologically hazardous shipping / handling restrictions. Cell size variation and differences between user-prepared cells make them unsuitable for specific instrument calibration controls. Furthermore, cell control material is often difficult to obtain when testing for rare diseases.

[0027] In contrast to polystyrene, the particles of the present disclosure exhibit cell-like autofluorescence and spectral profiles, allowing for more sensitive instrument calibration, better fluorescence compensation, and better overall experimental data analysis. The particles are also synthetically manufactured, allowing for high batch-to-batch precision without any of the drawbacks of using cell controls.

[0028] As shown in Figures 1A-1B and 2, polystyrene particles are fundamentally limited in the optical properties they can possess, such as forward and side scattering. This is primarily due to the fact that, in contrast to cells, such particles are opaque, and therefore side scattering is a direct function of particle size, as opposed to the complexity of the cell's interior. For example, Figure 1A(A) shows exemplary optical properties of engineered hydrogel particles of the present disclosure, where light from an excitation light source can interact with the internal structure of the engineered hydrogel particle to generate side scattering information about its internal structure. In contrast, Figures 1A(B) and 1B show exemplary optical properties of polystyrene beads, where light from an excitation light source does not interact with the internal structure of the polystyrene beads, and therefore the side scattering information obtained is limited. Furthermore, as shown in Figure 2, polystyrene beads have a side scattering profile that is three to four orders of magnitude different from that of real cells (e.g., target biological cells). Furthermore, polystyrene has high autofluorescence in many channels, even in the absence of fluorophores, resulting in reduced detector resolution (see, for example, Figures 7A-7C, discussed further below). In other instances, polystyrene exhibits low autofluorescence compared to cellular material, resulting in inaccurate staining index calculations, compensation, or spectral unmixing. This phenomenon makes it extremely difficult or impossible to accurately measure rare or poorly expressed biomarkers in samples. This also results in poor compensation performance in channels where polystyrene autofluoresces. Due to these limitations of polystyrene, users often must rely on purified cell lines to calibrate fluorescence intensity, fluorescence compensation, interlaser delay, sorting delay, size, and cellular complexity for immunophenotyping experiments. This is a lengthy, labor-intensive process that significantly increases the cost of flow cytometry validation and research pipelines. More importantly, these calibration cell lines introduce biological variation, causing variability in the interpretation of immunophenotyping data.

[0029] To utilize multiple fluorophores for a given biomarker phenotyping experiment, the fluorophores must be distinguishable on the cytometry instrument. The fluorescence profile of a given antibody, when bound to cells containing the same biomarker / antigen, can be used to compare it with other antibody-fluorophore combinations used in the same reagent "panel." Because using cells for fluorescence compensation is difficult, polystyrene beads are often used as a surrogate during fluorescence compensation setup. However, the background autofluorescence of polystyrene results in poor detector resolution, inaccurate compensation matrix calculations, background autofluorescence, and a poor lower detection threshold.

[0030] Embodiments of the present disclosure provide compositions comprising hydrogel particles having background fluorescence characteristics (e.g., autofluorescence) that are substantially similar to those of target cells (e.g., human cells) and overcome the various drawbacks of polystyrene discussed above. The hydrogel particles described herein can have a background spectral profile that is substantially similar to that of target cells. The inventors unexpectedly discovered that the fluorescence characteristics of hydrogel particles can be independently modulated by varying the composition of the hydrogel particles. Furthermore, the authors found that the background fluorescence characteristics of hydrogel particles can be modulated without affecting the particle's baseline optical properties (i.e., autofluorescence can be modulated independently of forward scatter (FSC) and side scatter (SSC)). This property enables the hydrogel to accurately mimic both the optical and autofluorescence characteristics of target cells measured by a cytometry device.

[0031] The present disclosure also provides a method of producing hydrogel particles, wherein the hydrogel particles have fluorescent properties substantially similar to those of target cells. The present disclosure also provides a method of producing hydrogel particles, wherein the hydrogel particles have predetermined optical or fluorescent properties. A method of calibrating a cytometry device for analysis of target cells is also provided, the method comprising: a) inserting into the device hydrogel particles having fluorescent properties substantially similar to those of target cells; and b) measuring the fluorescent properties of the hydrogel particles using the cytometry device, thereby calibrating the cytometry device for analysis of the target cells. Known cytometry devices include commercially available devices for performing flow cytometry, fluorescence-activated cell sorting (FACS), hematology, and high-content imaging. hydrogel

[0032] The hydrogel particles of the present disclosure include hydrogels. Hydrogels are materials containing a three-dimensional polymer network that swells in the presence of water and shrinks in the absence of water (or upon a decrease in the amount of water), but is insoluble in water. The swelling, i.e., absorption of water, is the result of the presence of hydrophilic functional groups attached to or dispersed within the polymer network. Crosslinks between adjacent polymers result in the water insolubility of these hydrogels. The crosslinks can be due to chemical (e.g., covalent) or physical (e.g., van der Waals forces, hydrogen bonds, ionic forces, etc.) bonds. While some in the polymer industry may refer to one or more of the polymeric materials described herein as "xerogels" in the dry state and "hydrogels" in the hydrated state, for purposes of this disclosure, the term "hydrogel" refers to the polymeric material whether dehydrated or hydrated. A particularly valuable feature of hydrogels is that the material retains its general shape whether dehydrated or hydrated. Thus, if the hydrogel has an approximately spherical shape in the dehydrated state, the hydrogel will be spherical in the hydrated state.

[0033] The disclosed hydrogels of the present disclosure, according to some embodiments, can comprise, by way of example, greater than about 30% water, greater than about 40% water, greater than about 50% water, greater than about 55% water, greater than about 60% water, greater than about 65% water, greater than about 70% water, greater than about 75% water, greater than about 80% water, or greater than about 85% water.

[0034] Synthetically prepared hydrogels can be prepared by polymerizing monomeric materials to form a backbone and crosslinking the backbone with a crosslinker. Common hydrogel monomers include lactic acid, glycolic acid, acrylic acid, 1-hydroxyethyl methacrylate, ethyl methacrylate, propylene glycol methacrylate, acrylamide, N-vinylpyrrolidone, methyl methacrylate, glycidyl methacrylate, glycol methacrylate, ethylene glycol, fumaric acid, and the like. Common crosslinkers include tetraethylene glycol dimethacrylate and N,N'-15 methylenebisacrylamide. In some embodiments, the hydrogel particles of the present disclosure are produced by polymerization of acrylamide.

[0035] In some embodiments, the hydrogel comprises a mixture of at least one monofunctional monomer and at least one difunctional monomer.

[0036] The monofunctional monomer may be a monofunctional acrylic monomer. Non-limiting examples of the monofunctional acrylic monomer include acrylamide, methacrylamide, N-alkylacrylamides such as N-ethylacrylamide, N-isopropylacrylamide, or N-tert-butylacrylamide, N-alkylmethacrylamides such as N-ethylmethacrylamide or N-isopropylmethacrylamide, N,N-dialkylacrylamides such as N,N-dimethylacrylamide and N,N-diethylacrylamide, and N-[(dialkylamino)alkyl]acrylamides such as N-[3-dimethylamino)propyl]acrylamides. N-[(dialkylamino)alkyl]methacrylamides, such as N-[3-dimethylamino)propyl]methacrylamide or N-[3-(diethylamino)propyl]methacrylamide; (dialkylamino)alkyl acrylates, such as 2-(dimethylamino)ethyl acrylate, 2-(dimethylamino)propyl acrylate, or 2-(diethylamino)ethyl acrylate; and (dialkylamino)alkyl methacrylates, such as 2-(dimethylamino)ethyl methacrylate.

[0037] A bifunctional monomer is any monomer that can be polymerized with a monofunctional monomer of the present disclosure to form a hydrogel as described herein that further comprises a second functional group that can participate in a second reaction, such as conjugation of a fluorophore.

[0038] In some embodiments, the difunctional monomer is selected from the group consisting of allyl alcohol, allyl isothiocyanate, allyl chloride, and allyl maleimide.

[0039] The difunctional monomer may be a difunctional acrylic monomer. Non-limiting examples of difunctional acrylic monomers are N,N'-methylenebisacrylamide, N,N'-methylenebismethacrylamide, N,N'-ethylenebisacrylamide, N,N'-ethylenebismethacrylamide, N,N'-propylenebisacrylamide, and N,N'-(1,2-dihydroxyethylene)bisacrylamide.

[0040] As described in U.S. Pat. No. 6,657,030, the entire contents of which are incorporated herein by reference, higher order branched and linear comonomers can be substituted into the polymer mixture to adjust the refractive index while maintaining polymer density.

[0041] In some embodiments, the hydrogel comprises a molecule that modulates the optical properties of the hydrogel. Molecules that can change the optical properties of the hydrogel are discussed further below.

[0042] Naturally occurring hydrogels useful in the present invention include a variety of polysaccharides available from natural sources such as plants, algae, fungi, yeast, marine invertebrates, and arthropods. Non-limiting examples include agarose, dextran, chitin, cellulosics, starch, derivatized starches, and the like, which generally have glucose repeating units as the majority of the polysaccharide backbone.

[0043] Polymerization of the hydrogel can be initiated by a persulfate. The persulfate can be any water-soluble persulfate. Non-limiting examples of water-soluble persulfates include ammonium persulfate and alkali metal persulfates. Alkali metals include lithium, sodium, and potassium. In some preferred embodiments, the persulfate is ammonium persulfate or potassium persulfate, more preferably ammonium persulfate.

[0044] Polymerization of the hydrogel can be accelerated by an accelerator. The accelerator can be a tertiary amine. The tertiary amine can be any water-soluble tertiary amine. Preferably, the tertiary amine is N,N,N',N'-tetramethylethylenediamine or 3-dimethylaminopropionitrile, more preferably N,N,N',N'-tetramethylethylenediamine (TEMED). Hydrogel particles

[0045] In one embodiment, the hydrogel particles of the present disclosure comprise a hydrogel and are produced by polymerizing droplets (see FIG. 5). Microfluidic methods for producing multiple droplets, including fluid droplets and rigidified droplets, are known and are described in U.S. Patent Application Publication No. 2011 / 0218123 and U.S. Patent No. 7,294,503, the entire contents of each of which are incorporated herein by reference. Such methods provide multiple droplets comprising a first fluid and substantially surrounded by a second fluid, where the first and second fluids are substantially immiscible (e.g., droplets containing an aqueous liquid are substantially surrounded by an oil-based liquid). In another embodiment, the hydrogel particles are produced by precipitation or chemical polymerization. In another embodiment, the hydrogel particles are produced by membrane emulsification. In another embodiment, the hydrogel particles are formed via piezoelectric dispersion.

[0046] A plurality of fluidic droplets (e.g., prepared using a microfluidic device) may be polydisperse (e.g., having a range of different sizes), or in some cases, the fluidic droplets may be monodisperse or substantially monodisperse, e.g., having a uniform distribution of diameters, e.g., such that no more than about 10%, about 5%, about 3%, about 1%, about 0.03%, or about 0.01% of the droplets have an average diameter greater than about 10%, about 5%, about 3%, about 1%, about 0.03%, or about 0.01% of the average diameter. As used herein, the average diameter of a population of droplets refers to the arithmetic mean of the diameters of the droplets.

[0047] Accordingly, the present disclosure provides a population of hydrogel particles comprising a plurality of hydrogel particles, wherein the population of hydrogel particles is substantially monodisperse.

[0048] The term microfluidic refers to a device, apparatus, or system comprising at least one fluid channel having a cross-sectional dimension of less than 1 mm and a ratio of length to maximum cross-sectional dimension perpendicular to the channel of at least about 3: 1. Microfluidic devices comprising microfluidic channels are particularly well suited for the preparation of multiple monodisperse droplets.

[0049] Non-limiting examples of microfluidic systems that may be used with the present invention include U.S. Patent Application Publication No. 2006 / 0163385 ("Forming and Control of Fluidic Species"), U.S. Patent Application Publication No. 2005 / 0172476 ("Method and Apparatus for Fluid Dispersion"), U.S. Patent Application Publication No. 2007 / 000342 ("Electronic Control of Fluidic Species"), WO 2006 / 096571 ("Method and Apparatus for Forming Multiple Emulsions"), U.S. Patent Application Publication No. 2007 / 0054119 ("Systems and Methods of Forming Particles"). Particles”), WO 2008 / 121342 (“Emulsions and Techniques for Formation”), and WO 2006 / 078841 (“Systems and Methods for Forming Fluidic Droplets Encapsulated in Particles Such as Colloidal Particles”), the entire contents of each of which are incorporated herein by reference in their entirety.

[0050] The droplet size is related to the microfluidic channel size. The microfluidic channel can be any size, for example, less than about 5 mm, or less than 2 mm, or less than about 1 mm, or less than about 500 μm, less than about 200 μm, less than about 100 μm, less than about 60 μm, less than about 50 μm, less than about 40 μm, less than about 30 μm, less than about 25 μm, less than about 10 μm, less than about 3 μm, less than about 1 μm, less than about 300 nm, less than about 100 nm, less than about 30 nm, or less than about 10 nm in the largest dimension perpendicular to the fluid flow.

[0051] Droplet size can be adjusted by adjusting the relative flow rates, in some embodiments, the droplet diameter is equal to or within about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the channel width.

[0052] The dimensions of the hydrogel particles of the present disclosure are substantially similar to the droplets from which they are formed. Thus, in some embodiments, the hydrogel particles have a diameter of less than about 1 μm, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 600, 800 μm, or less than 1000 μm. In some embodiments, the hydrogel particles have a diameter greater than about 1 μm, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 600, 800 μm, or greater than 1000 μm. In typical embodiments, the hydrogel particles have a diameter in the range of 5 μm to 100 μm.

[0053] In some embodiments, the hydrogel particles of the present disclosure are spherical.

[0054] In some embodiments, hydrogel particles of the present disclosure have material modulus properties (eg, elasticity) that more closely resemble those of target cells compared to polystyrene beads of the same diameter.

[0055] In some embodiments, the hydrogel particles of the present disclosure do not include agarose. optical properties Passive and non-passive optical properties (e.g., fluorescence properties)

[0056] The three primary modes of deconvolution for flow cytometry are the two passive optical properties of the particle (forward scatter (FSC), or RI, corresponding to refractive index; and side scatter (SSC)) and fluorescence, which is a non-passive optical property (i.e., a feature imparted by a molecule that is not a component of the base polymer, such as a fluorophore, fluorescent dye, or quantum dot), typically measured using antibodies with conjugated fluorophores and representative of biomarkers present on the surface of a given cell type. Therefore, compositions that enable the disclosed hydrogel particles to mimic specific cell types with respect to these three modes are useful for providing synthetic, robust calibrators for flow cytometry.

[0057] In some embodiments, the refractive index (RI) of the disclosed hydrogel particles is greater than about 1.10, greater than about 1.15, greater than about 1.20, greater than about 1.25, greater than about 1.30, greater than about 1.35, greater than about 1.40, greater than about 1.45, greater than about 1.50, greater than about 1.55, greater than about 1.60, greater than about 1.65, greater than about 1.70, greater than about 1.75, greater than about 1.80, greater than about 1.85, greater than about 1.90, greater than about 1.95, greater than about 2.00, greater than about 2.10, greater than about 2.20, greater than about 2.30, greater than about 2.40, greater than about 2.50, greater than about 2.60, greater than about 2.70, greater than about 2.80, or greater than about 2.90.

[0058] In some embodiments, the refractive index (RI) of the disclosed hydrogel particles is less than about 1.10, less than about 1.15, less than about 1.20, less than about 1.25, less than about 1.30, less than about 1.35, less than about 1.40, less than about 1.45, less than about 1.50, less than about 1.55, less than about 1.60, less than about 1.65, less than about 1.70, less than about 1.75, less than about 1.80, less than about 1.85, less than about 1.90, less than about 1.95, less than about 2.00, less than about 2.10, less than about 2.20, less than about 2.30, less than about 2.40, less than about 2.50, less than about 2.60, less than about 2.70, less than about 2.80, or less than about 2.90.

[0059] The SSC of the disclosed hydrogel particles is most meaningfully measured relative to the SSC of target cells, hi some embodiments, the disclosed hydrogel particles have an SSC within 30%, within 25%, within 20%, within 15%, within 10%, within 5%, or within 1% of the SSC of target cells as measured by a cytometry device.

[0060] The FSC of the disclosed hydrogel particles is most meaningfully measured relative to the FSC of target cells. In some embodiments, the disclosed hydrogel particles have an FSC within 30%, 25%, 20%, 15%, 10%, 5%, or 1% of the FSC of target cells as measured by a cytometry device.

[0061] The FSC can be adjusted for a hydrogel by incorporating high refractive index molecules into the hydrogel. Preferred high refractive index molecules include colloidal silica, alkyl acrylates, and alkyl methacrylates. Thus, in some embodiments, the hydrogel particles of the present disclosure comprise alkyl acrylates and / or alkyl methacrylates.

[0062] The alkyl acrylate or alkyl methacrylate may contain 1 to 18, 1 to 8, or 2 to 8 carbon atoms in the alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, 2-ethylhexyl, heptyl, or octyl groups. The alkyl group may be branched or linear.

[0063] High refractive index molecules may also include vinylarenes such as styrene and methylstyrene optionally substituted on the aromatic ring with alkyl groups such as methyl, ethyl, or tert-butyl, or with halogens such as chlorostyrene.

[0064] In some embodiments, FSC is modulated by adjusting the water content present during hydrogel formation.

[0065] FSC is related to particle volume and therefore can be adjusted by varying particle diameter as described herein.

[0066] SSCs can be engineered by encapsulating nanoparticles within the hydrogel to mimic organelles within target cells. In some embodiments, the hydrogel particles of the present disclosure comprise one or more types of nanoparticles selected from the group consisting of polymethyl methacrylate (PMMA) nanoparticles, polystyrene (PS) nanoparticles, and silica nanoparticles. Functionalization of hydrogel particles

[0067] The hydrogel particles can be functionalized to mimic the optical and fluorescent properties of labeled cells. In some embodiments, the hydrogel particles comprise bifunctional monomers, and the functionalization of the hydrogel particles occurs via the bifunctional monomers. In typical embodiments, the functionalized hydrogel particles comprise free amine groups.

[0068] The hydrogel particles can be functionalized with any of the fluorescent dyes known in the art, including those listed in The Molecular Probes® Handbook—A Guide to Fluorescent Probes and Labeling Technologies, which is incorporated herein by reference in its entirety. Functionalization can be mediated by compounds containing free amine groups, such as allylamine, which can be incorporated into the hydrogel particles during the formation process.

[0069] Non-limiting examples of known fluorescent dyes include 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimidyl ester, 5-(and-6)-carboxyeosin; 5-carboxyfluorescein; 6-carboxyfluorescein; 5-(and-6)-carboxyfluorescein; 5-carboxyfluorescein-bis-(5-carboxymethoxy-2-nitrobenzyl)ether, -alanine-carboxamide, or succinimidyl ester; 5-carboxyfluorescein succinimidyl ester. ;6-Carboxyfluorescein succinimidyl ester;5-(and-6)-Carboxyfluorescein succinimidyl ester;5-(4,6-Dichlorotriazinyl)aminofluorescein;2',7'-Difluorofluorescein;Eosin-5-isothiocyanate;Erythrosine 5-isothiocyanate;6-(Fluorescein-5-carboxamido)hexanoic acid or succinimidyl ester;6-(Fluorescein-5-(and-6)-carboxamido)hexanoic acid or succinimidyl ester;Fluorescein Fluorescein-5-EX succinimidyl ester; fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate; OregonGreen® 488 carboxylic acid, or succinimidyl ester; OregonGreen® 488 isothiocyanate; OregonGreen® 488-X succinimidyl ester; OregonGreen® 500 carboxylic acid; OregonGreen® 500 carboxylic acid, succinimidyl ester, or triethylammonium Salts; OregonGreen® 514 carboxylic acid; OregonGreen® 514 carboxylic acid or succinimidyl ester; Rhodamine Green™ carboxylic acid, succinimidyl ester or hydrochloride salt; Rhodamine Green™ carboxylic acid, trifluoroacetamide or succinimidyl ester; Rhodamine Green™-X succinimidyl ester or hydrochloride salt; RhodolGreen™ carboxylic acid, N,0-bis-(trifluoroacetyl) or succinimidyl ester;Bis-(4-carboxypiperidinyl)sulfone rhodamine or di(succinimidyl ester); 5-(and -6) carboxynaphthofluorescein, 5-(and -6) carboxynaphthofluorescein succinimidyl ester; 5-carboxyrhodamine 6G hydrochloride; 6-carboxyrhodamine 6G hydrochloride, 5-carboxyrhodamine 6G succinimidyl ester; 6-carboxyrhodamine 6G succinimidyl ester; 5-(and -6)-carboxyrhodamine 6G succinimidyl ester Ester;5-Carboxy-2',4',5',7'-tetrabromosulfonefluorescein succinimidyl ester or bis-(diisopropylethylammonium) salt;5-Carboxytetramethylrhodamine;6-Carboxytetramethylrhodamine;5-(and -6)-Carboxytetramethylrhodamine;5-Carboxytetramethylrhodamine succinimidyl ester;6-Carboxytetramethylrhodamine succinimidyl ester;5-(and -6)-Carboxytetramethylrhodamine Lissamine succinimidyl ester; 6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimidyl ester; 6-carboxy-X-rhodamine succinimidyl ester; 5-(and -6)-carboxy-X-rhodamine succinimidyl ester; 5-carboxy-X-rhodamine triethylammonium salt; Lissamine™ rhodamine B sulfonyl chloride; Malachite Green; Isothiocyanate; NANOGOLD® mono(sulfosuccinimidyl ester) QSY® 21 carboxylic acid or succinimidyl ester; QSY® 7 carboxylic acid or succinimidyl ester; Rhodamine Red™-X succinimidyl ester; 6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoic acid; succinimidyl ester; tetramethylrhodamine-5-isothiocyanate; tetramethylrhodamine-6-isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Red® sulfonyl; Texas Red® sulfonyl chloride; Texas Red®-X STP ester or sodium salt;Texas Red®-X succinimidyl ester; Texas Red®-X succinimidyl ester; and X-rhodamine-5-(and -6)-isothiocyanate.

[0070] Other examples of fluorescent dyes include the BODIPY® dyes commercially available from Invitrogen, including, but not limited to, BODIPY® FL; BODIPY® TMR STP Ester; BODIPY® TR-X STP Ester; BODIPY® 630 / 650-X STP Ester; BODIPY® 650 / 665-X STP Ester; 6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester; 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3,5-dipropionic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid; 4, 4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid succinimidyl ester;4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid;4,4-Difluoro-5,7-dimethyl-4-bora-3a,4-diaza-s-indacene-3-propionic acid succinimidyl ester;4,4- Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid; sulfosuccinimidyl ester or sodium salt; 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoic acid; 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza- s-Indacene-3-propionyl)amino)hexanoic acid or succinimidyl ester; N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)cysteic acid, succinimidyl ester or triethylammonium salt; 6-4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid; 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester;4,4-Difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid; Succinimidyl ester; 6-((4,4-Difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoic acid or succinimidyl ester; 4,4-Difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester ;4,4-Difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester;6-(((4,4-Difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryloxy)acetyl)aminohexanoic acid or succinimidyl ester;4,4-Difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid;4,4-Difluoro- 5-Styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid;Succinimidyl ester;4,4-Difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-propionic acid;4,4-Difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-propionic acid;4,4-Difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-propionic acid succinimidyl ester;4,4-Difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene indacen-3-propionic acid succinimidyl ester; 6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-indacen-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimidyl ester; and 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacen-3-yl)styryloxy)acetyl)aminohexanoic acid or succinimidyl ester;

[0071] Fluorescent dyes may also include Alexa fluor dyes commercially available from Invitrogen, including, but not limited to, Alexa Fluor® 350 carboxylic acid, Alexa Fluor® 430 carboxylic acid, Alexa Fluor® 488 carboxylic acid, Alexa Fluor® 532 carboxylic acid, Alexa Fluor® 546 carboxylic acid, Alexa Fluor® 555 carboxylic acid, Alexa Fluor® 568 carboxylic acid, Alexa Fluor® 594 carboxylic acid, Alexa Fluor® 633 carboxylic acid, Alexa Fluor® 647 carboxylic acid, Alexa Fluor® 660 carboxylic acid, and Alexa Fluor® 680 carboxylic acid. Fluorescent dyes of the present invention may also be cyanine dyes commercially available from Amersham-Pharmacia Biotech, including, but not limited to, Cy3 NHS ester, Cy5 NHS ester, Cy5.5 NHS ester, and Cy7 NHS ester.

[0072] Tandem dyes, such as those containing PE-Cy5 or other combinations, can also be effectively utilized in the present disclosure due to their low autofluorescence. Typically, polystyrene autofluorescence interferes with the fluorescence resonance energy transfer (FRET) signal required to utilize tandem or polymeric dyes. target cell

[0073] The hydrogel particles of the present disclosure behave similarly to target cells in procedures such as staining and analysis by flow cytometry or FACS.

[0074] In some embodiments, the target cell is an immune cell. Non-limiting examples of immune cells include B lymphocytes, also called B cells, T lymphocytes, also called T cells, natural killer (NK) cells, lymphokine-activated killer (LAK) cells, monocytes, macrophages, neutrophils, granulocytes, mast cells, platelets, Langerhans cells, stem cells, dendritic cells, peripheral blood mononuclear cells, tumor-infiltrating (TIL) cells, genetically modified immune cells including hybridomas, drug-modified immune cells, and derivatives, precursors, or progenitors of any of the cell types listed herein.

[0075] In some embodiments, target cells include all cells of a particular class of cells that share common characteristics. For example, target cells can be lymphocytes, including NK cells, T cells, and B cells. Target cells can be activated lymphocytes.

[0076] In some embodiments, the target cell is a primary cell, a cultured cell, a cell line, a normal cell, a transformed cell, an infected cell, a stably transfected cell, a transiently transfected cell, a proliferating cell, or a terminally differentiated cell.

[0077] In one embodiment, the target cell is a primary neuron cell. Various neurons can be target cells. By way of non-limiting example, the target cell can be a primary neuron, an established neuron; a transformed neuron; a stably transfected neuron; or a motor or sensory neuron.

[0078] In other embodiments, the target cells are selected from the group consisting of primary lymphocytes, monocytes, and granulocytes.

[0079] Target cells can be virtually any type of cell, including prokaryotic and eukaryotic cells.

[0080] Suitable prokaryotic target cells include, but are not limited to, bacteria such as E. coli, various Bacillus species, and extremophilic bacteria such as thermophilic bacteria.

[0081] Suitable eukaryotic target cells include, but are not limited to, fungi such as yeast and filamentous fungi, including species of Saccharomyces, Aspergillus, Trichoderma, and Neurospora; plant cells, including those of corn, sorghum, tobacco, rapeseed, soybean, cotton, tomato, potato, alfalfa, sunflower, and the like; and animal cells, including fish, birds, and mammals. Suitable fish cells include, but are not limited to, those derived from salmon, trout, tilapia, tuna, carp, flounder, halibut, swordfish, cod, and zebrafish species. Suitable bird cells include, but are not limited to, those derived from chicken, duck, quail, pheasant, and turkey, as well as other jungle foul or game birds. Suitable mammalian cells include, but are not limited to, horse, cow, buffalo, deer, sheep, rabbit, rodents such as mice, rats, hamsters and guinea pigs, goats, pigs, primates, marine mammals including dolphins and whales, and cell lines, such as human cell lines of any tissue or stem cell type, and cells from stem cells, including pluripotent and non-pluripotent, and non-human zygotes.

[0082] Suitable cells also include cell types involved in a wide variety of disease states, even in non-disease states. Thus, suitable eukaryotic cell types include, but are not limited to, all types of tumor cells (e.g., melanoma, myeloid leukemia, lung, breast, ovarian, colon, kidney, prostate, pancreatic, and testicular cancer), cardiomyocytes, dendritic cells, endothelial cells, epithelial cells, lymphocytes (T cells and B cells), mast cells, eosinophils, endothelial cells, macrophages, natural killer cells, erythrocytes, hepatocytes, leukocytes including monocytes, stem cells (for use in screening for differentiation and dedifferentiation factors), such as hematopoietic stem cells, neural stem cells, skin stem cells, lung stem cells, kidney stem cells, liver stem cells, and muscle stem cells, osteoclasts, chondrocytes and other connective tissue cells, keratinocytes, melanocytes, hepatocytes, kidney cells, and adipocytes. In certain embodiments, the cells are primary disease-state cells, such as primary tumor cells. Suitable cells also include known research cells, including but not limited to Jurkat T cells, NIH3T3 cells, CHO, COS, etc. See the ATCC Cell Line Catalog, expressly incorporated herein by reference.

[0083] In some embodiments, the target cells are tumor microvesicles or tumor giant vesicles. Tumor microvesicles, also known as tumor-secreted microvesicles or tumor-secreted exosomes, can be found in the circulating blood and may have immunosuppressive activity. Tumor microvesicles typically range in size from 30 to 200 nm in diameter. Larger tumor microvesicles, sometimes called tumor giant vesicles, can range in size from 3 to 10 μm in diameter. [Example]

[0084] Example 1: Production of hydrogel particles Photomasks for UV lithography were procured from CADart Services Inc. and designed using AutoCad (AutoDesk, Inc.). SU-8 photoresist (Microchem, Inc.) was photocrosslinked on 4-inch silicon wafers using a collimated UV light source (OAI, Inc.) to create masters for microfluidic device fabrication. PDMS (polydimethylsiloxane, Sigma-Aldrich, Inc.) was prepared and molded using standard published methods for soft lithography and microfluidic device fabrication (see McDonald JC et al., 2000, Electrophoresis 21:27-40).

[0085] Droplets were formed using a flow-focusing geometry, in which two oil channels focused the central flow of the aqueous monomer solution, separating droplets in a water-in-oil emulsion. Fluorocarbon oil (Novec 7500, 3M, Inc.) was used as the outer continuous phase liquid for droplet formation. To stabilize the droplets before polymerization, a surfactant (Krytox 157 FSH ammonium carboxylate, DuPont) was added to the oil phase at 0.5% (w / w). Basic polyacrylamide gel particles were fabricated using a central phase of aqueous monomer solution containing N-acrylamide (1–20% (w / v)), a crosslinker (N,N'-bisacrylamide, 0.05–1% (w / v)), an accelerator, and ammonium persulfate (1% (w / v)). To induce hydrogel particle polymerization after droplet formation, an accelerator (N,N,N',N'-tetramethylethylenediamine 2% (vol %)) was added to the oil phase.

[0086] Several comonomers were added to basic gel formulations to add functionality. In one example, an aryl acrylate was added to modulate the autofluorescence properties of the particles. In another example, polystyrene nanoparticles were added to the hydrogel matrix at low concentrations to modulate the autofluorescence properties of the particles. Fluorescence properties were also modulated by adjusting the crosslink density of the particles and manipulating the kinetics of the crosslinking and curing process (e.g., by varying the temperature, time, and / or concentration of one or more accelerators). The comonomers, nanoparticle additives, and crosslink density of the basic gel formulations were modulated to affect the fluorescence and spectral properties of the particles and create formulation models that mimic cell-like background optical responses. Specifically, the type of chemical side groups present on various comonomers incorporated into the gel matrix, as well as the comonomer concentration, additives, and crosslink density of the core polymer, affect the fluorescence and spectral properties of the particles.

[0087] Stoichiometric multiplexing of hydrogel particles was achieved by utilizing comonomers containing chemically orthogonal side groups (e.g., amine, carboxyl, maleimide, epoxide, alkyne) for secondary labeling.

[0088] We generated droplets at an average rate of 5 kHz and collected them in a fluorocarbon oil phase. After completing the polymerization at 50 °C for 30 min, the resulting hydrogel particles were washed from the oil into an aqueous solution. Example 2: Multidimensional tuning of hydrogel particle optical properties

[0089] As shown in Figure 3, the autofluorescence properties of hydrogel particles (unlike polystyrene beads) can be tuned in multiple dimensions to match the cell-like background autofluorescence / spectral profile of specific cell types. Independent tuning of autofluorescence and forward scattering can be achieved by modulating / selectively modifying the comonomer composition, nanoparticle additive composition, and / or crosslinking density of the hydrogel particles. Cells are deconvoluted using optical parameters, such as FSC and SSC, or a combination of secondary markers. Unlike polystyrene beads, which have limited size and side scattering (shown in Figure 1A(B) and Figure 1B), hydrogel particles can be tuned to precisely match the SSC and FSC of specific cell types. Figure 1A(A) shows that the cells and engineered hydrogels described herein are translucent, allowing internal features to be elucidated via side scattering (SSC). In contrast, as shown in Figure 1A(B), polystyrene beads are opaque and have a defined side scattering determined by their diameter. Hydrogel particles can be further functionalized with stoichiometrically tuned ratios of specific chemical side groups and secondary labels, allowing for precise matching of any cell type without the biological noise of fixed cell lines (see Figure 4). Specifically, as shown in Figure 4, the multiplexing capabilities of functionalized hydrogels allow for the addition of antigens and other biomarkers to the hydrogel base polymer, adding an additional "cell-like" dimension to the product. Example 3: Comparison of engineered hydrogel particles with polystyrene particles and cells

[0090] Hydrogel particles were formed using the method described above and measured in all fluorescence channels of a Beckman Coulter Cytoflex instrument. 5 μm polystyrene beads (BD Biosciences) were measured in parallel. Cells obtained from a commercial supplier were run in phosphate-buffered saline and measured on a Beckman Coulter Cytoflex instrument. Figure 6 shows plots of intensity versus wavelength / channel demonstrating the principle of fluorescence compensation. Specifically, Figure 6 illustrates the concept of fluorescence spillover and compensation. As shown in Figure 6, the primary detection channel (A) exhibits the highest intensity for the model fluorophore, while channels B and C exhibit spillover or residual emission signal from a single fluorophore. Such values ​​can be subtracted from the measured fluorescence signal when combined with other fluorophores emitting in these channels to calculate a more accurate, or "true," fluorescence signal intensity.

[0091] 7A-7C facilitate a comparison of the fluorescence signatures between lymphocyte cells, polystyrene beads, and the engineered hydrogel autofluorescent hydrogels of the present disclosure ("FlowCytes"). Each plot represents a standard fluorescence detection channel and an exemplary antigen or biological target named in that detection channel during a common experiment. The channels are as follows: Channel FL1-A - ThiozolOrange-A (DNA-binding photosensitizer) Channel FL2-A-PerCP-A (PerCP-conjugated antibody, where PerCP is a peridinin-chlorophyll-protein, i.e., fluorescent complex) Channel FL3-A-CD4 APC-A (cluster of differentiation (CD) 4 allophycocyanin (APC) antibody) Channel FL4-A-APC-A700-A (conjugated antibody) Channel FL5-A-APC-A750-A (conjugated antibody) Channel FL6-A-BV421-1 (brilliant violet 421 antibody conjugate) Channel FL7-A-BV510-A (brilliant violet 421 antibody conjugate) Channel FL8-A - Violet610-A fluorescent nanoparticle dye Channel FL9-A - Violet660-A fluorescent nanoparticle dye Channel FL10-A-PE-A (phycoerythrin antibody) Channel FL11-A-ECO-A Channel FL12-A-7AAO-A (7-aminoactinomycin D)

[0092] As shown in Figures 7A-7C, flow cytometers exhibit more cell-like autofluorescence (i.e., their associated autofluorescence signatures more closely resemble those of lymphocyte cells) compared to polystyrene beads. This allows for a larger dynamic range of measurements on the same instrument, enabling more accurate fluorescence compensation. For example, Figure 7C shows that flow cytometers have lower autofluorescence in the ultraviolet and violet spectrum, making them more cell-like (i.e., more similar to lymphocyte cells than polystyrene beads). Furthermore, flow cytometers have a relatively high signal-to-noise ratio, which facilitates better detection of poorly expressed or "dimmed" biomarkers by reducing the noise floor and increasing the dynamic range of a given detector. The hydrogels described herein also enable the use of synthetic bead products with fluorescent dyes that excite or emit in the violet and ultraviolet ranges—properties that cannot be matched by current polystyrene-based products.

[0093] 8A is a spectral profile of lymphocytes stained with Alexa 700-modified Ab, according to one embodiment, and FIG. 8B is a spectral profile of hydrogel particles (flow cytometer) of the present disclosure stained with Alexa 700-modified Ab. As shown in FIGS. 8A-8B, the stained flow cytometers show 2It has a cell-like spectral signature with a peak-to-peak match of .gtoreq.1.

[0094] 9 is a flow diagram illustrating a method for calibrating a cytometry device for analysis of target cells, according to some embodiments. As shown in FIG. 9, method 900 optionally includes, at 902, obtaining or generating hydrogel particles having at least one background fluorescent characteristic and / or at least one spectral characteristic substantially similar (e.g., within 10%) to corresponding at least one fluorescent characteristic and / or at least one spectral characteristic of a target cell (e.g., a human cell). At 904, method 900 includes inserting at least one hydrogel particle (e.g., optionally a plurality of hydrogel particles in an aqueous medium or solution) into the cytometry device. The method also includes, at 906, measuring the fluorescent characteristic of the hydrogel particle using the cytometry device.

[0095] Figure 10 is a flow diagram illustrating a process for calibration and calculation of fluorescence compensation and spectral unmixing using hydrogels of the present disclosure. As shown in Figure 10, process 1000 includes, at 1008, modifying hydrogel particles to attach antibody-fluorophore conjugates or DNA-binding dyes (e.g., anti-kappa light chain antibodies) to the hydrogel particles. At 1010, individual reagents are attached, and the hydrogel particles are inserted into a cytometry device and their fluorescence and / or spectral properties are measured. Then, at 1012, a fluorescence compensation matrix and / or spectral unmixing table is calculated for a plurality of individual fluorophores.

[0096] 11A-11D are bar graphs showing a comparison of cell staining, according to some embodiments, with hydrogel compensation beads and known (polystyrene-based) bead products. In all cases shown, the hydrogel beads of the present disclosure exhibit more cell-like characteristics, resulting in superior compensation and spectral unmixing performance. FIG. 11A lists the staining index and degradation performance of the hydrogel compensation beads compared to known products. As can be seen from FIG. 11A, the staining index of the hydrogel compensation beads of the present disclosure is more cell-like than the known compensation bead products. FIG. 11B lists the mean fluorescence intensity (MFI) of the stained hydrogel compared to known compensation products. As can be seen from FIG. 11B, the stained hydrogel compensation beads have a more cell-like MFI compared to the known compensation bead products. FIG. 11C lists the background autofluorescence of the hydrogel compared to known compensation products. As can be seen from FIG. 11C, the unstained hydrogel compensation beads have a more cell-like background autofluorescence across a wide range of channels compared to the known compensation bead products. Figure 11D describes the spillover performance of the hydrogel compared to known compensation products. Figure 11D shows that spillover of the fluorescent channel is superior for the hydrogel compensation beads of the present disclosure compared to known compensation bead products.

[0097] FIG. 12 is a chart illustrating two exemplary methods for tuning the autofluorescence of hydrogel particles: (1) modulating the percentage of resonant comonomer additive or (2) varying the crosslink density of the hydrogel, according to some embodiments. FIG. 12 compares the autofluorescence of polystyrene and cell controls with the autofluorescence of these exemplary hydrogel particles. As can be seen from the table on the left of FIG. 12, the hydrogel particles prepared with 5% resonant comonomer additive have cell-like autofluorescence (1050), while the polystyrene control has undesirably high autofluorescence (9781). As can be seen from the table on the right of FIG. 12, the hydrogel particles prepared with 10% crosslink density have cell-like autofluorescence (1104), while the polystyrene control has undesirably high autofluorescence (9781).

[0098] In some embodiments, the composition comprises an aqueous solution and hydrogel particles suspended in the aqueous solution. The hydrogel particles have at least one of a background autofluorescence substantially similar to that of target cells or a spectral profile substantially similar to that of target cells. These specific properties are engineered using a combination of comonomer additives, tuned curing kinetics (which are affected by, and can therefore be tuned by, time, temperature, and chemical accelerators), and low-concentration nanoparticle additives. These properties (autofluorescence and spectral profile) are characterized using non-passive optical excitation channels and are distinguished from passive optical signatures (such as SSC and FSC).

[0099] The hydrogel particles can also have an SSC within 10% of the SSC of the target cells as measured by a cytometry device.The hydrogel particles can also have an FSC within 10% of the FSC of the target cells as measured by a cytometry device.

[0100] The hydrogel particles can also have a refractive index greater than about 1.15, or greater than about 1.3, or greater than about 1.7.

[0101] The hydrogel particles can also have a diameter of less than about 100 μm, or less than about 10 μm, or less than about 1 μm.

[0102] In some embodiments, the hydrogel particles contain a polymeric nanoparticle additive.

[0103] In some embodiments, the hydrogel particles are chemically functionalized, for example, the hydrogel particles can include free amine groups.

[0104] In some embodiments, the hydrogel particles comprise allylamine.

[0105] In some embodiments, the target cell is an immune cell.

[0106] In some embodiments, the hydrogel particles are produced by polymerizing the droplets.

[0107] In some embodiments, hydrogel particles are produced by polymerizing droplets, and the hydrogel particles are then modified by conjugating or attaching a fluorophore / fluorescent dye. The modified hydrogel particles can have a fluorescence profile that matches (e.g., is substantially similar to or within 10% of) the fluorescence profile of the target cells.

[0108] In some embodiments, the population of hydrogel particles comprises a plurality of hydrogel particles, and each hydrogel particle from the plurality of hydrogel particles has at least one of background autofluorescence or a spectral profile substantially similar to the background autofluorescence or spectral profile of a target cell. The population of hydrogel particles can be substantially monodisperse. In some such embodiments, 10% or less of the hydrogel particles have an average diameter greater than about 10% of the average diameter of the population of hydrogel particles.

[0109] In some embodiments, the method includes calibrating a cytometry device for analysis of target cells by inserting at least one hydrogel particle (e.g., a plurality of hydrogel particles, optionally in an aqueous medium or solution) into the cytometry device. The at least one hydrogel particle has at least one background fluorescence characteristic or spectral characteristic substantially similar to at least one background fluorescence characteristic (e.g., autofluorescence) or spectral characteristic of the target cells. The method also includes measuring at least one characteristic (e.g., a characteristic related to calibration) of the hydrogel particle using the cytometry device. The at least one characteristic can include one or more of inter-laser delay, fluorescence response, sort timing, or fluorescence compensation. The method also optionally includes adjusting either fluorescence compensation or spectral unmixing based on the measured characteristic. Spectral unmixing is the process of decomposing the spectral signature of a mixed pixel into a set of endmembers and their corresponding abundances. The described compensation and spectral unmixing calculations using cell-like reagents allow for multiplexing of a wide range of fluorophores by reducing noise and increasing cell-like accuracy for a given fluorophore. In some embodiments, the method also includes conjugating a fluorophore-containing reagent to the hydrogel particles to form a complex prior to inserting the hydrogel particles into a cytometry device, measuring at least one property of the complex, and calculating fluorescence compensation or spectral unmixing based on the at least one measured property. Optionally, the method also includes using the modified hydrogel particles to assess the viability of target cells.

[0110] In some embodiments, the hydrogel particles are modified to bind to antibodies that are linked to conjugated fluorophores (eg, fluorescent dyes).

[0111] In some embodiments, the hydrogel particles are modified hydrogel particles that have been modified to bind at least one of an intercalating nucleic acid labeling reagent or an amine-reactive nucleic acid labeling reagent.

[0112] The hydrogel particles can have an SSC within 10% of the SSC of the target cells as measured by a cytometry device. Alternatively or additionally, the hydrogel particles can have an FSC within 10% of the FSC of the target cells as measured by a cytometry device.

[0113] In some embodiments, the hydrogel particles can have a refractive index greater than about 1.15, or greater than about 1.3, or greater than about 1.7.

[0114] In some embodiments, the hydrogel particles can have a diameter of less than about 100 μm, or less than about 10 μm, or less than about 1 μm.

[0115] In some embodiments, the hydrogel particles include a polymeric nanoparticle additive.

[0116] In some embodiments, the hydrogel particles are chemically functionalized hydrogel particles.

[0117] In some embodiments, the hydrogel particles comprise free amine groups.

[0118] In some embodiments, the hydrogel particles comprise allylamine.

[0119] In some embodiments, the target cell is an immune cell.

[0120] In some embodiments, the method also includes polymerizing the droplets to form hydrogel particles.

[0121] In some embodiments, the hydrogel particles are modified by conjugating or attaching one of a fluorophore or fluorescent dye, where the modified hydrogel particles match the fluorescence or spectral profile of the cells.

[0122] In some embodiments, a method includes calculating a correction value for cytometric measurements of target cells and correcting the cytometric measurements of the target cells based on the correction value. Calculating the correction value for the cytometric measurements of the target cells includes inserting first hydrogel particles into a cytometry device at a first time. The first hydrogel particles have at least one background fluorescence characteristic or spectral characteristic substantially similar to at least one background fluorescence characteristic or spectral characteristic of the target cells. The at least one characteristic of the first hydrogel particles is measured using the cytometry device. The calculating also includes inserting second hydrogel particles into the cytometry device at a second time, different from the first time, and measuring at least one characteristic of the second hydrogel particles using the cytometry device. The calculating also includes comparing the measured at least one characteristic of the first hydrogel particles with the measured at least one characteristic of the second hydrogel particles to determine the correction value.

[0123] In some embodiments, the method includes calculating a plurality of adjustment values ​​for the cytometric measurement of the target cells and correcting the cytometric measurement of the target cells based on the plurality of adjustment values. Calculating the plurality of adjustment values ​​for the cytometric measurement of the target cells includes inserting into a cytometry device two hydrogel particles, the first hydrogel particle from hydrogel particles having at least one background fluorescent characteristic or spectral characteristic substantially similar to at least one background fluorescent characteristic or spectral characteristic of the target cells, and the second hydrogel particle from hydrogel particles configured to bind to or pre-bound to a reagent, the reagent being a reagent that generates at least one of a fluorescent signal that differs from the background fluorescent characteristic and a spectral signal that differs from the spectral characteristic. Calculating a plurality of adjustment values ​​for the cytometric measurement of the target cells also includes measuring at least one property of the first hydrogel particles and at least one property of the second hydrogel particles using the cytometry device, and comparing the measured at least one property of the first hydrogel particles with the measured at least one property of the second hydrogel particles to determine fluorescent overlap with the at least one additional reagent and spectral overlap with the at least one additional reagent. The cytometric measurement of the target cells is then corrected based on the plurality of adjustment values ​​(e.g., including or based on the fluorescent overlap with the at least one additional reagent and / or the spectral overlap with the at least one additional reagent).

[0124] Although shown and described herein as being used in the context of cytometry device calibration and cytometry measurement correction, the cell-like hydrogel particles described herein can also be used in other applications to improve their performance and / or accuracy. For example, additional applications compatible with the cell-like hydrogel particles of the present disclosure include, but are not limited to, the following: (1) setting the lower limit of detection ("LLOD") of an instrument (including, but not limited to, a flow cytometer, hematology analyzer, cytometer, or image-based cytometer) to determine the true signal-to-noise ratio for weakly or poorly expressed biomarkers; (2) adjusting the gain of a photomultiplier tube ("PMT") to capture cell-like fluorescence linearity; (3) calculating mean fluorescence intensity ("MFI"); and (4) instrument setup and quality control ("QC") for fluorescence detection (active, as opposed to passive, optical properties).

[0125] While various particular embodiments have been illustrated and described, it will be understood that various modifications can be made without departing from the spirit and scope of the invention.

[0126] As used throughout this specification and the appended claims, the following terms and phrases are intended to have the following meanings:

[0127] The indefinite articles "a" and "an" and the definite article "the" are intended to include both the singular and the plural, unless the context clearly indicates otherwise.

[0128] "At least one" and "one or more" are used interchangeably to mean that an article may contain one or more than one of the listed elements.

[0129] Unless otherwise indicated, all values ​​expressing quantities, ratios, and numerical properties of ingredients, reaction conditions, and the like used in the specification and claims are intended to be modified in all instances by the term "about."

[0130] As used herein, the terms "about" and "approximately" generally mean plus or minus 10% of the stated value, for example, about 250 μm would include 225 μm to 275 μm, and about 1,000 μm would include 900 μm to 1,100 μm.

[0131] In this disclosure, references to singular items should be understood to include plural items, and vice versa, unless otherwise stated or clear from the context. Grammatical conjunctions are intended to represent any and all disjunctive and conjunctive combinations of joined clauses, sentences, words, etc., unless otherwise stated or clear from the context. Thus, the term "or" should generally be understood to mean "and / or," etc. The use of any and all examples or exemplary language (such as "for example," "such as," "including," etc.) provided herein is intended merely to further clarify the embodiments and does not limit the scope of the embodiments or claims.

Claims

1. 1. A method of calibrating a cytometry device for analysis of target cells, comprising: a) inserting into the cytometry device hydrogel particles having background fluorescence characteristics substantially similar to the background fluorescence characteristics of the target cells; b) measuring the fluorescence of the hydrogel particles using the cytometry device, thereby calibrating the cytometry device; wherein the background fluorescence property of the hydrogel particles is caused by a resonant comonomer, by encapsulated autofluorescent polymer nanoparticles, or by modulated hydrogel crosslink density.

2. Before step (a), binding a fluorophore-containing reagent to the hydrogel particles to form a complex; After step (a) but before step (b), calculating a fluorescence correction or spectral unmixing based on the at least one measured fluorescence of the hydrogel particles; The method of claim 1 further comprising:

3. 10. The method of claim 1, wherein the hydrogel particles are modified to bind to an antibody that binds a conjugated fluorophore.

4. 4. The method of claim 3, wherein the conjugated fluorophore is a fluorescent dye.

5. 5. The method of claim 1, wherein the hydrogel particles are modified hydrogel particles that have been modified to bind to at least one of an intercalating nucleic acid labeling reagent or an amine-reactive labeling reagent.

6. 6. The method of claim 5, further comprising assessing the viability of the target cells using the modified hydrogel particles.

7. The method of any one of claims 1 to 6, wherein the hydrogel particles have a refractive index greater than about 1.

15.

8. The method of any one of claims 1 to 6, wherein the hydrogel particles have a refractive index greater than about 1.

3.

9. The method of any one of claims 1 to 6, wherein the hydrogel particles have a refractive index greater than about 1.

7.

10. The method of any one of claims 1 to 9, wherein the hydrogel particles have a diameter of less than about 100 μm.

11. The method of any one of claims 1 to 9, wherein the hydrogel particles have a diameter of less than about 10 μm.

12. The method of any one of claims 1 to 9, wherein the hydrogel particles have a diameter of less than about 1 μm.

13. The method of any one of claims 1 to 12, wherein the hydrogel particles contain a polymeric nanoparticle additive.

14. The method of any one of claims 1 to 13, wherein the hydrogel particles are chemically functionalized hydrogel particles.

15. The method of any one of claims 1 to 14, wherein the hydrogel particles comprise free amine groups.

16. The method of any one of claims 1 to 15, wherein the hydrogel particles comprise allylamine.

17. The method of any one of claims 1 to 16, wherein the target cell is an immune cell.

18. The method of any one of claims 1 to 17, further comprising polymerizing the droplets to form the hydrogel particles.

19. 10. The method of claim 1, wherein the hydrogel particles are hydrogel particles modified by conjugating or attaching one of a fluorophore or a fluorescent dye.

20. 20. The method of claim 19, wherein the modified hydrogel particles match the fluorescence or spectral profile of a cell.

21. 21. The method of any one of claims 1 to 20, wherein the measuring step comprises measuring a property selected from the group consisting of inter-laser delay, fluorescence response, sort timing, and fluorescence compensation.

22. Calculating a plurality of adjustment values ​​for cytometric measurements of target cells, the adjustment values ​​comprising: inserting first hydrogel particles and second hydrogel particles into the cytometry device, the first hydrogel particles having at least one background fluorescent characteristic or spectral characteristic substantially similar to at least one background fluorescent characteristic or spectral characteristic of the target cells; inserting the second hydrogel particles, wherein the second hydrogel particles are either configured to bind to or are pre-bound to a reagent, the reagent being a reagent that generates at least one of a fluorescent signal that is different from the background fluorescent characteristic or a spectral signal that is different from the spectral characteristic; measuring at least one property of the first hydrogel particles and at least one property of the second hydrogel particles using the cytometry device; comparing the measured at least one property of the first hydrogel particles with the measured at least one property of the second hydrogel particles to determine a fluorescence overlap with at least one additional reagent and a spectral overlap with the at least one additional reagent; Calculating by, and modifying the cytometric measurement of the target cells based on the plurality of adjustment values; A method comprising:

23. a) background fluorescence characteristics that are substantially similar to the background fluorescence characteristics of the target cells; and b) optical properties substantially similar to the optical properties of said target cells 1. A composition comprising hydrogel particles having: the optical properties include forward scattering and / or side scattering; the background fluorescence properties of the hydrogel particles are caused by resonant comonomers, by encapsulated autofluorescent polymer nanoparticles, or by modulated hydrogel crosslink density; composition.

24. 24. The composition of claim 23, wherein the hydrogel particles comprise a monomer selected from hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinylpyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glycerol methacrylate (GMA), glycol methacrylate, ethylene glycol, fumaric acid, and any combination thereof.

25. 24. The composition of claim 23, wherein the hydrogel particles are configured to bind to an antibody that binds to a conjugated fluorophore.

26. 26. The composition of claim 25, wherein the conjugated fluorophore is a fluorescent dye.

27. 27. The composition of any one of claims 23 to 26, wherein the hydrogel particles are modified hydrogel particles modified to bind to at least one of an intercalating nucleic acid labeling reagent or an amine-reactive labeling reagent.

28. The composition of any one of claims 23 to 27, wherein the hydrogel particles have a refractive index greater than about 1.

15.

29. The composition of any one of claims 23 to 27, wherein the hydrogel particles have a refractive index greater than about 1.

3.

30. The composition of any one of claims 23 to 27, wherein the hydrogel particles have a refractive index greater than about 1.

7.

31. The composition of any one of claims 23 to 30, wherein the hydrogel particles have a diameter of less than about 100 μm.

32. The composition of any one of claims 23 to 30, wherein the hydrogel particles have a diameter of less than about 10 μm.

33. The composition of any one of claims 23 to 30, wherein the hydrogel particles have a diameter of less than about 1 μm.

34. The composition of any one of claims 23 to 33, wherein the hydrogel particles contain a polymeric nanoparticle additive.

35. The composition of any one of claims 23 to 34, wherein the hydrogel particles are chemically functionalized hydrogel particles.

36. The composition of any one of claims 23 to 35, wherein the hydrogel particles comprise free amine groups.

37. The composition of any one of claims 23 to 36, wherein the hydrogel particles comprise allylamine.

38. The composition of any one of claims 23 to 37, wherein the target cell is an immune cell.

39. 39. The composition of any one of claims 23 to 38, wherein the hydrogel particles are hydrogel particles modified by conjugating or attaching one of a fluorophore or a fluorescent dye.

40. 40. The composition of any one of claims 23 to 39, wherein the hydrogel particles are modified to match the fluorescence profile of the target cells.

41. The composition of any one of claims 23 to 40, wherein the target cell is a human cell.

42. A composition comprising hydrogel particles having (1) background fluorescence characteristics substantially similar to the background fluorescence characteristics of target cells, and (2) optical properties substantially similar to the optical properties of said target cells, said hydrogel particles being modified to bind to at least one of an intercalating nucleic acid labeling reagent or an amine-reactive labeling reagent, wherein said background fluorescence characteristics of said hydrogel particles are provided by a resonant comonomer, by encapsulated autofluorescent polymeric nanoparticles, or by modulated hydrogel crosslink density.

43. A composition comprising hydrogel particles having (1) background fluorescence characteristics substantially similar to those of target cells, and (2) optical properties substantially similar to those of said target cells, wherein said hydrogel particles have a refractive index greater than about 1.15, and wherein said background fluorescence characteristics of said hydrogel particles are provided by a resonant comonomer, by encapsulated autofluorescent polymer nanoparticles, or by modulated hydrogel crosslink density.

44. A composition comprising hydrogel particles having (1) background fluorescence characteristics substantially similar to those of target cells, and (2) optical properties substantially similar to those of said target cells, wherein said hydrogel particles have a refractive index greater than about 1.3, and wherein said background fluorescence characteristics of said hydrogel particles are provided by a resonant comonomer, by encapsulated autofluorescent polymer nanoparticles, or by modulated hydrogel crosslink density.

45. A composition comprising hydrogel particles having (1) background fluorescence characteristics substantially similar to those of target cells, and (2) optical properties substantially similar to those of said target cells, wherein said hydrogel particles have a refractive index greater than about 1.7, and wherein said background fluorescence characteristics of said hydrogel particles are provided by a resonant comonomer, by encapsulated autofluorescent polymer nanoparticles, or by modulated hydrogel crosslink density.

46. A composition comprising hydrogel particles having (1) background fluorescence characteristics substantially similar to the background fluorescence characteristics of target cells, and (2) optical properties substantially similar to the optical properties of said target cells, said hydrogel particles comprising a polymeric nanoparticle additive, wherein said background fluorescence characteristics of said hydrogel particles are provided by a resonant comonomer, by encapsulated autofluorescent polymeric nanoparticles, or by modulated hydrogel crosslink density.

47. A composition comprising hydrogel particles having (1) background fluorescence characteristics substantially similar to those of target cells, and (2) optical properties substantially similar to those of the target cells, wherein the hydrogel particles comprise free amine groups, and the background fluorescence characteristics of the hydrogel particles are provided by a resonant comonomer, by encapsulated autofluorescent polymer nanoparticles, or by modulated hydrogel crosslink density.

48. A composition comprising hydrogel particles having (1) background fluorescence characteristics substantially similar to the background fluorescence characteristics of target cells, and (2) optical properties substantially similar to the optical properties of said target cells, wherein said hydrogel particles comprise allylamine, and wherein said background fluorescence characteristics of said hydrogel particles are provided by a resonant comonomer, by encapsulated autofluorescent polymer nanoparticles, or by modulated hydrogel crosslink density.

49. A composition comprising hydrogel particles having (1) background fluorescence characteristics substantially similar to the background fluorescence characteristics of target cells, and (2) optical properties substantially similar to the optical properties of said target cells, said hydrogel particles being modified by conjugating or attaching one of a fluorophore or fluorescent dye, wherein said background fluorescence characteristics of said hydrogel particles are provided by a resonant comonomer, by encapsulated autofluorescent polymer nanoparticles, or by modulated hydrogel crosslink density.

50. A composition comprising hydrogel particles having (1) background fluorescence characteristics substantially similar to the background fluorescence characteristics of target cells, and (2) optical properties substantially similar to the optical properties of the target cells, wherein the hydrogel particles are modified to match the fluorescence profile of the target cells, and the background fluorescence characteristics of the hydrogel particles are provided by a resonant comonomer, by encapsulated autofluorescent polymer nanoparticles, or by modulated hydrogel crosslink density.

51. A composition comprising hydrogel particles having background fluorescence characteristics substantially similar to those of target cells, wherein the background fluorescence characteristics of the hydrogel particles are provided by a resonant comonomer, by encapsulated autofluorescent polymer nanoparticles, or by modulated hydrogel crosslink density.

52. 23. The method of claim 22, wherein the background fluorescence property of the first hydrogel particles is caused by a resonant comonomer, by encapsulated autofluorescent polymer nanoparticles, or by modulated hydrogel crosslink density.

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