Systems and methods for generating immobilized partition matrices and spatially fixing and analyzing targets

Abstract

This disclosure provides for devices, methods, and systems for immobilizing partitions of a partition matrix, where partitions are rapidly generated from a sample, and for immobilizing a set of targets within a matrix, where examples of the matrix are optically clear. Embodiments of methods involve partition generation within a collecting container at a high rate (e.g., of at least 1 million partitions per minute, etc.), each of the partitions including an aqueous mixture. Embodiments of methods also involve stabilizing positions of targets within a matrix within a collecting container and performing reactions within the matrix at a temperature below the melting temperature of the matrix. Upon immobilization, readout of signals from targets within such partitions and / or the matrix can still be achieved in accordance with various assays.

Description

Atty. DocketNo.: 43161-64728 / WO (003WO)SYSTEMS AND METHODS FOR GENERATING IMMOBILIZED PARTITION MATRICES AND SPATIALLY FIXING AND ANALYZING TARGETS1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application Nos.: 63 / 725,548, filed November 27, 2024. and 63 / 830,353, filed June 25. 2025, the entire disclosures of which are hereby incorporated by reference in their entireties.2. BACKGROUND

[0002] In biotechnology and other applications, target distribution and partitioning technologies play a significant role in achieving the ability to conduct microscale and nanoscale analyses of targets (e.g., of single cells, of single molecules, of other analytes, of particles, etc.). Distributing targets in space, in a manner that allows individual targets to be detected, and / or dispersing samples across partitions, in a consistent and reliable manner has utility in relation to various assays in cell and molecular biology, with respect to digital analyses (e.g., digital polymerase chain reaction), quantitative analyses, and other bioassays. Isolated and / or independent reaction environments, including those provided in a partitioned- format, can further greatly reduce the sample and process fluid volume required, reducing costs associated with sample processing. Results returned from such assays can be used for clinical and non-clinical characterizations of various conditions.

[0003] Currently-available technologies are also unable to provide immobilized or positionally-stabilized targets and / or partitions that can be optically interrogated in bulk format and transported for interrogation or further processing, in a robust and cost-effective manner.

[0004] As such, there is a need in the field of sample processing for new and improved partitioning technologies, and for technologies for fixing positions of targets (e.g., targets of a sample) and detecting individual targets. The disclosure provided herein covers such technologies.3. SUMMARY

[0005] Currently, methods and systems for distributing and spatially fixing targets of a sample and / or distributing targets of samples across partitions in a rapid and consistent manner, in a manner where partition contents are immobilized and stably isolated without leakage or merging of contents of adjacent partitions, in a manner where the spatially-fixedAtty. DocketNo.: 43161-64728 / WO (003WO) targets and / or bulk arrangement of partitions can be transported, and in a manner that enables readout of signals from the spatially -fixed targets, in 3D format, and / or partitions, are severely limited. Commercially available platforms typically isolate targets and / or conduct partitioning by using microfluidic devices involving complex setups, and / or have limited ability to distinctly detect signals corresponding to targets when the targets are distributed across three-dimensions in space (e.g., in bulk format within a medium). However, such platforms may be costly, distribute and / or partition samples at a slow rate and / or with high levels of sample loss contributing to poor dead volume performance, are labor-intensive, or can cause sample contamination. Thus, there is a need in the field of sample processing to create new and useful systems and methods for fixing positions of targets (e.g., targets of a sample) and detecting individual targets, and / or for generation of and / or stabilization of partitions.

[0006] Accordingly, this disclosure describes embodiments, variations, and examples of systems, methods, and compositions for generating partitions in bulk format, and immobilizing generated partitions. This disclosure also describes embodiments, variations, and examples of systems, methods, and compositions for stabilizing positions of targets and detecting individual targets (e.g., within an optically clear matrix).

[0007] In some embodiments of a matrix as disclosed herein, the matrix is a partition matrix.

[0008] An aspect of the disclosure provides embodiments, variations, and examples of a partition matrix, wherein a set of partitions of the partition matrix is immobilized within a closed container with a polymer material, and wherein the partition matrix with the polymer material has a level of optical clarity7above a threshold level of clarity. The polymer material can include linear polymer structures and / or branched polymer structures. The polymer material can be a thermoplastic or a thermosetting polymer material. In some embodiments, the polymer material comprises a thermoplastic polymer material. In some embodiments, the thermoplastic polymer material comprises agarose. In some embodiments, the polymer material is in a form of a polymer material-containing solution. In some embodiments, the polymer material -containing solution comprises 0.02 to 3% w / v% of agarose. In some embodiments, the polymer material comprises a thermosetting polymer material. The polymer material can be a crosslinked material or a material that is not crosslinked.

[0009] In some embodiments, the polymer material can be combined with one or more additional components that provide functionality7to the polymer, where the additional component(s) can diffuse or otherwise be transmitted into the partitions of the partition matrix (e.g., in order to interact with targets captured in the partitions of the partition matrix).Atty. DocketNo.: 43161-64728 / WO (003WO)Embodiments of the additional component(s) can include one or more of: chemical probes, affinity ligands, enzymatic components, dyes (e.g., SYBR Green, hydrolysis probes, nonhydrolysis probes, cell viability-associated dyes, etc.), sequence-specific capture agents (e.g., antisense oligonucleotides, LNA-modified probes, PNA clamps, morpholino oligomers, etc.), CRISPR guide RNAs, molecular beacons, enzymes (e.g., polymerases, reverse transcriptases, ligases, nickases, recombinases, strand-displacing polymerases, etc ), small molecules (e.g., bis-intercalators (e.g., YOYO-1), minor-groove binders (e.g., netropsin, distamycin), G- quadruplex stabilizers (e g., TMPyP4, BRACO-19), etc ), cell-penetrant viability dyes (calcein-AM, SYTO dyes), membrane-impermeant exclusion dyes (7-AAD, TO-PRO-3), lectins, aptamers, antibody-conjugated fluorophores, and metabolic reporters (resazurin, tetrazolium salts), metal-chelating probes, click-chemistry tags, barcoding oligos, lysis reagents, permeabilization reagents, fixing reagents, and / or other materials.

[0010] In relation to optical clarity, the threshold level can be a level greater than 60% transmissivity of light, greater than 65% transmissivity of light, greater than 70% transmissivity of light, greater than 75% transmissivity of light, greater than 80% transmissivity of light, greater than 85% transmissivity of light, greater than 90% transmissivity of light, greater than 95 % transmissivity of light, greater than 99% transmissivity of light, or greater. In some embodiments, the optical clarity threshold level can be a level of at least 60% transmissivity of light, at least 65% transmissivity of light, at least 70% transmissivity of light, at least 75% transmissivity of light, at least 80% transmissivity of light, at least 85% transmissivity of light, at least 90% transmissivity of light, at least 95% transmissivity' of light, at least 99% transmissivity of light, or greater. In some embodiments, the optical clarity threshold level is at least 60% transmissivity of light, at least 70% transmissivity of light, or at least 80% transmissivity of light. In some embodiments, the optical clarity threshold level is at least 80% transmissivity’ of light.However, variations of the partition matrix can have optical clarity below 60% transmissivity of light. In some embodiments, the partition matrix with the polymer material has optical clarity above the threshold level of clarity without use of refractive index matching between the partition matrix and the polymer material, and wherein the threshold level is at least 60% transmissivity of light, at least 65% transmissivity’ of light, at least 70% transmissivity of light, at least 75% transmissivity of light, at least 80% transmissivity of light, at least 85% transmissivity of light, at least 90% transmissivity of light, at least 95% transmissivity of light, at least 99% transmissivity of light, or another suitable threshold level. In some embodiments, the partition matrix with the polymer material has optical clarity above theAtty. DocketNo.: 43161-64728 / WO (003WO) threshold level of clarity without use of refractive index matching between the partition matrix and the polymer material, and wherein the threshold level is at least 60% transmissivity of light, at least 70% transmissivity of light, at least 80% transmissivity of light, or another suitable threshold level. In examples, the partition matrix with the polymer material has optical clarity above the threshold level of clarity without use of refractive index matching between the partition matrix and the polymer material, and wherein the threshold level is at least 80% transmissivity of light or another suitable threshold level.

[0011] An aspect of the disclosure provides embodiments, variations, and examples of a device for generating partitions from a sample fluid, wherein the device includes: a first substrate defining a reservoir comprising a reservoir inlet and a reservoir outlet; a membrane coupled to the reservoir outlet and comprising a distribution of holes; and a supporting body comprising an opening configured to retain a collecting container in alignment with the reservoir outlet. During operation, the first substrate can be coupled with the supporting body and enclose the collecting container, with the reservoir outlet aligned with and / or seated within the collecting container. During operation, the reservoir can contain a sample, where application of a force to the device or sample fluid generates a plurality of partitions of from sample fluid transmission into the collecting container at a high rate (e.g., of at least 50,000 partitions / minute, of at least 100,000 partitions / minute, of at least 200,000 partitions / minute, of at least 300,000 partitions / minute, of at least 400,000 partitions / minute, of at least 500,000 partitions / minute, of at least 600,000 partitions / minute, of at least 700,000 partitions / minute. of at least 800,000 partitions / minute, of at least 900,000 partitions / minute, of at least 1 million partitions / minute, of at least 2 million partitions / minute, of at least 3 million partitions / minute, etc.), where the partitions are stabilized in position / immobilized (e.g., in a close-packed format, in equilibrium stationary positions) within the collecting container. Notably, the partitions are stable across a wide range of temperatures (e.g., 1 °C through 95°C, greater than 95 °C, less than 1 °C) relevant to various digital analyses and other bioassays. Once immobilized, the partitions within the closed collecting container can form a fixed partition matrix. In some embodiments, the fixed partition matrix is a gel.

[0012] In embodiments, variations, and examples, the membrane includes a distribution of holes having low- density’ (e.g., significantly lower than that typical for filtration applications involving porous membranes).

[0013] In embodiments, variations, and examples, the device can include a set of reservoirs (e.g., at the first substrate), a set of membranes at outlets of the set of reservoirs, and a supporting body for a set of collecting containers, in order to provide parallel processing ofAtty. DocketNo.: 43161-64728 / WO (003WO) multiple samples and / or combination of sample processing materials during the partitioning process.

[0014] In embodiments, variations, and examples, the device can include a spacer configured to separate the membrane(s) further from base surfaces or liquid interfaces within the collecting container(s), thereby enabling operation modes in which partitions emerging from the membrane pass through air or another initial fluid phase prior to arriving at respective equilibrium positions.

[0015] An aspect of the disclosure provides embodiments, variations, and examples of a method for rapidly generating partitions (e.g., partitions from a sample fluid, partitions of an emulsion) within a collecting container at a high rate, each of the plurality of partitions including an aqueous mixture for a digital analysis (e.g., of nucleic acid material, of protein material, of amino acid material, of other analytes described herein), wherein upon generation, the plurality of partitions is stabilized in position (e.g., in a close-packed format, at equilibrium stationary positions, etc.) within a continuous phase (e g., as an emulsion having a bulk morphology defined by the collecting container). The continuous phase can be aqueous, and each of the plurality of partitions can be surrounded by an immiscible film or other immiscible layer that is immiscible with the sample fluid and the continuous phase. In aspects, partition generation can be executed by driving the sample fluid through a distribution of holes of a membrane, where the applied force can be one or more of centrifugal, associated with applied pressure, magnetic, or otherwise physically applied.

[0016] As such, a set of partitions of the partition matrix can contain a set and / or distribution of targets (e.g., nucleic acid targets, protein targets). In some embodiments, the set and / or distribution of targets comprises a set and / or distribution of nucleic acid targets. In some embodiments, the set and / or distribution of targets comprises a set and / or distribution of protein targets. In variations, each of the set of partitions contains at most one target of the set and / or distribution of targets. In variations, each of the set of partitions contains at most one nucleic acid target of the set and / or distribution of nucleic acid targets. In variations, each of the set of partitions contains at most one protein target of the set and / or distribution of protein targets.

[0017] An aspect of the disclosure provides embodiments, variations, and examples of a method for fixing positions of a partition matrix by immobilizing the partition matrix within a collecting container upon: transmitting a polymer material in a flow state into the collecting container and over the partition matrix, wherein the partition matrix has an optical clarity above a threshold level of clarity’; and transitioning the polymer material to a set state withinAtty. DocketNo.: 43161-64728 / WO (003WO) the collecting container, while maintaining optical clarity of the partition matrix above the threshold level of clarity. Threshold levels of clarity are described herein. In some embodiments, the partition matrix comprises a set of aqueous partitions generated from a sample, wherein the set of aqueous partitions is stabilized in position in a close-packed format within an aqueous continuous phase, and wherein each of the set of aqueous partitions comprises a thin fdm (or other layer) that is immiscible with the aqueous continuous phase. In some embodiments, each partition of a set of partitions of the partition matrix is surrounded by the polymer material. In some embodiments, the polymer material is provided at a surface of the partition matrix as a cap. In some embodiments, the polymer material comprises a thermoplastic polymer material. In some embodiments, the thermoplastic polymer material comprises agarose. In some embodiments, the polymer material is in a form of a polymer material-containing solution. In some embodiments, the polymer materialcontaining solution comprises 0.02 to 3% w / v% of agarose. In some embodiments, the polymer material comprises a thermosetting polymer material. In some embodiments, transmitting the polymer material in the flow state comprises heating the polymer material prior to transmission into the collecting container. In some embodiments, transitioning the polymer material to the set state comprises centrifuging the collecting container in coordination with cooling of the polymer material within the collecting container. In some embodiments, transitioning the polymer material to the set state comprises cross-linking the polymer material. In some embodiments, the set of partitions of the partition matrix contains a set of targets. In some embodiments, each of the set of partitions contains at most one target of the set of targets. In some embodiments, the set of targets comprises a set of nucleic acid targets. In some embodiments, the set of targets comprises a set of protein targets. In some embodiments, the partition matrix with the polymer material has optical clarity above the threshold level of clarity without use of refractive index matching between the partition matrix and the polymer material. In some embodiments, the threshold level is at least 80% transmissivity of light.

[0018] In some embodiments, the polymer material can be combined with one or more additional components that provide functionality to the polymer, where the additional component(s) can diffuse or otherwise be transmitted into the partitions of the partition matrix (e.g., in order to interact with targets captured in the partitions of the partition matrix). Embodiments of the additional component(s) can include one or more of: chemical probes, affinity ligands, enzymatic components, dyes (e.g., SYBR Green, hydrolysis probes, nonhydrolysis probes, cell viability-associated dyes, etc.), sequence-specific capture agents (e.g..Atty. DocketNo.: 43161-64728 / WO (003WO) antisense oligonucleotides, LNA-modified probes, PNA clamps, morpholino oligomers, etc.), CRISPR guide RNAs, molecular beacons, enzymes (e.g., polymerases, reverse transcriptases, ligases, nickases, recombinases, strand-displacing polymerases, etc.), small molecules (e.g., bis-intercalators (e.g., YOYO-1), minor-groove binders (e.g., netropsin, distamycin), G- quadruplex stabilizers (e g., TMPyP4, BRACO-19), etc ), cell-penetrant viability dyes (calcein-AM, SYTO dyes), membrane-impermeant exclusion dyes (7-AAD, TO-PRO-3), lectins, aptamers, antibody-conjugated fluorophores, and metabolic reporters (resazurin, tetrazolium salts), metal-chelating probes, click-chemistry tags, barcoding oligos, lysis reagents, permeabilization reagents, fixing reagents, and / or other materials.

[0019] In embodiments, variations, and examples, generated partitions can form a fixed partition matrix, with contents of individual partitions of the partition matrix stabilized in position within a collecting container. The partition matrix can be a viscous fluid, a shearthickening fluid, a gel (e.g., a gel having individual discrete partitions), or another fluid having a surface. The partition matrix can be transitioned to a solidified state (e.g., as a solid), where partitions of the partition matrix are immobilized and do not move, even when a force is applied to the partition matrix and / or the partition matrix is transported.

[0020] In relation to a single-tube workflow in which the collecting container remains closed (e.g., the collecting container has no outlet, there is no flow out of the collecting container, to avoid sample contamination), method(s) can further include transmitting heat to and from the partition matrix within the closed collecting container (e.g.. according to an assay protocol). In relation to generation of emulsions having suitable clarity (e.g., with or without refractive index matching), method(s) can further include transmission of signals from individual partitions from the partition matrix for readout (e.g., by an optical detection platform, by another suitable detection platform).

[0021] Where method(s) include transmitting heat to and from the partition matrix, within the closed container, the partitions are stable across a wide range of temperatures (e.g., 1 °C through 95 °C, greater than 95 °C, less than 1 °C) relevant to various digital analyses and other bioassays, where the partitions remain consistent in morphology’ and remain unmerged with adjacent partitions.

[0022] Examples of partition generation methods can include generating a high number of partitions (e.g., greater than 100,000 partitions, greater than 500,000 partitions, greater than 1 million partitions, greater than 2 million partitions, greater than 3 million partitions, greater than 4 million partitions, greater than 5 million partitions, greater than 6 million partitions, greater than 7 million partitions, greater than 8 million partitions, greater than 9 millionAtty. DocketNo.: 43161-64728 / WO (003WO) partitions, greater than 10 million partitions, greater than 15 million partitions, greater than 20 million partitions, greater than 25 million partitions, greater than 30 million partitions, greater than 40 million partitions, greater than 50 million partitions, greater than 100 million partitions, etc.) from the sample, for transmission into a collecting container having a volumetric capacity (e.g., less than 50 microliters, from 50 through 100 microliters and greater, etc.), where resultant partitions have a characteristic dimension (e.g., from 1-50 micrometers, from 10-30 micrometers, etc.) that is relevant for digital analyses, single cell capture, target detection, individual molecule partitioning, or other applications.

[0023] The disclosure provides for systems, devices, and methods that enable digital analyses across a wide dynamic range that is 10-100 times greater than that of existing technologies, depending upon application of use. In examples related to nucleic acid counting, the disclosure provides for systems, devices, and methods that can have a dynamic range from 1 through 100 million, due to the high number of uniform partitions generated from which signals can be read, and due to the ability7to partition with low occupancy (e.g., less than 20% occupancy, less than 10% occupancy, less than 9% occupancy, less than 8% occupancy, less than 7% occupancy, less than 6% occupancy, less than 5% occupancy, etc.) of partitions by targets.

[0024] In specific applications, partitioning devices and methods described herein can perform: detection and counting of nucleic acid molecules via amplification of individual nucleic acid molecule captured within a partition followed by detection of optically detectable signals (e.g., amplification by polymerase chain reaction (PCR) methods, by isothermal methods such as loop-mediated isothermal amplification (LAMP), by recombinase polymerase amplification (RPA), by helicase dependent amplification (HD A), by strand displacement amplification (SDA), by nicking enzyme amplification (NEAR), by transcription mediated amplification (TMA), by RNaseH mediated amplification, by whole genome amplification (WGA) using phi29, by rolling circle amplification, etc.) on purified DNA, cDNA, RNA, oligonucleotide tagged antibodies / proteins / small molecules, or directly from lysate (e.g.. blood lysate); fluorescent in situ hybridization (FISH) with fluorescently tagged nucleic acids (e.g., PNA, LNA, DNA, RNA, etc.) or an indirect in situ hybridization approach using DIG or biotin, where the signal is later amplified by conjugation of an antibody to alkaline phosphatase or a peroxidase to produce a change in color detected by one or more substrates (e.g., nitroblue tetrazolium (NBT), 5-bromo-4-chloro-3-indolyl-phosphate (BCIP), HNPP, etc.); an in vitro transcription or translation assay whereby a colorimetric or fluorescent reporter is used for detection; partition PCR applied to samples derived fromAtty. DocketNo.: 43161-64728 / WO (003WO) single cells (e.g., prokaryotes, eukary otes), organelles, viral particles, and exosomes; enumeration of protein or peptide molecules (e.g.. by proximity ligation assays, etc.); sequencing applications (e.g., single molecule sequencing applications): monitoring or detection of products (e.g., proteins, chemicals) released from single cells (e.g., interleukin released from immune cells); monitoring cell survival and / or division for single cells; monitoring or detection of enzymatic reactions involving single cells; antibiotic resistance screening for single bacteria; enumeration of pathogens in a sample (e.g., in relation to infections, sepsis, in relation to environmental and food samples, etc.); enumeration of heterogeneous cell populations in a sample; enumeration of individual cells or viral particles (e.g., by encapsulating cells in partitions with species-specific antibodies coupled with enzymes that react with substrate components in the partition to produce signals, etc.); monitoring of viral infections of a single host cell; liquid biopsies and companion diagnostics; prenatal diagnosis of genetic disorders (e.g., aneuploidy, genetically inherited diseases) such as with cell-free nucleic acids, fetal cells, or samples containing mixtures of fetal and maternal cells; detection of cancer forms from various biological samples (e.g., detection of cancer from cell-free nucleic acids, tissue biopsies, biological fluids, feces); detection of markers (e.g., genetic markers) relevant to the field of oncology; detection and / or monitoring of minimal residual diseases; monitoring responses to therapies; detection or prediction of rejection events of transplanted organs; linkage analysis; detection of titer characteristics (e.g., viral titer characteristics); detection of integrity’; detection of full vs. empty capsid markers; other diagnostics associated with other health conditions; other characterizations of statuses of other organisms; and other suitable applications.

[0025] In specific applications, the systems, devices, and methods for partitioning in a single tube workflow can perform processes that achieve goals of digital PCR, quantitative PCR, and next generation sequencing, using a single platform.

[0026] In embodiments, the target material analyzed according to digital analysis and / or other bioassay techniques can include one or more of: nucleic acid material (e.g., DNA, RNA, miRNA, etc.), protein material, amino acid material, other small molecules, other single analytes, other multi-analytes, and / or other suitable target material of a sample. In embodiments, the sample can include or otherw ise be derived from: whole tissue structures, tissue portions (e.g., histological tissue slices, formalin-fixed paraffin-embedded (FFPE) tissue, frozen tissue, biopsied tissues, fresh frozen plasma, seeded natural scaffolds, seeded synthetic scaffolds, etc.), organs, whole organisms, organoids, cell suspensions (e.g.. frozen cell suspensions that are separated prior to processing w i th the system, cell suspensionsAtty. DocketNo.: 43161-64728 / WO (003WO) retained in a medium / hydrogel medium, etc ), nuclei suspensions, other suspensions, single cells, organelles, sub-organelle structures, intra-organelle components, viruses, microorganisms, and other samples.

[0027] An additional aspect of the present disclosure provides for a method comprising: generating a plurality of partitions within a collecting container at a rate of at least 1 million partitions per minute, each of the plurality of partitions comprising an aqueous mixture for a digital analysis of partitioned target material.

[0028] In some embodiments, a method for generating an immobilized partition matrix within a collecting container comprises: generating a plurality' of partitions within a collecting container at a rate of at least 1 million partitions / minute, each of the plurality of partitions including an aqueous mixture for a digital analysis; stabilizing the plurality of partitions as a partition matrix in a close-packed format within a continuous phase, within a region of the collecting container; transmitting a polymer material in a flow state into the collecting container and over the partition matrix; and transitioning the polymer material to a set state within the collecting container, wherein the partition matrix with the polymer material has optical clarity above a threshold level of clarity without use of refractive index matching between the partition matrix and the polymer material, wherein the threshold level is at least 80% transmissivity7of light, and wherein each partition of the plurality' of partitions contains at most one target of a set of targets

[0029] In some embodiments, the polymer material used for the method(s) described can be combined with one or more additional components that provide functionality to the polymer, where the additional component(s) can diffuse or otherwise be transmitted into the partitions of the partition matrix (e g., in order to interact with targets captured in the partitions of the partition matrix). Embodiments of the additional component(s) can include one or more of: chemical probes, affinity ligands, enzy matic components, dyes (e.g., SYBR Green, hydrolysis probes, non-hydrolysis probes, cell viability-associated dyes, etc.), sequencespecific capture agents (e.g., antisense oligonucleotides, LNA-modified probes, PNA clamps, morpholino oligomers, etc ), CRISPR guide RNAs, molecular beacons, enzymes (e.g., polymerases, reverse transcriptases, ligases, nickases, recombinases, strand-displacing polymerases, etc ), small molecules (e.g., bis-intercalators (e.g., YOYO-1), minor-groove binders (e.g., netropsin, distamycin), G-quadruplex stabilizers (e.g., TMPyP4, BRACO-19), etc.), cell-penetrant viability dyes (calcein-AM, SYTO dyes), membrane-impermeant exclusion dyes (7-AAD, TO-PRO-3), lectins, aptamers, antibody-conjugated fluorophores, and metabolic reporters (resazurin, tetrazolium salts), metal-chelating probes, click-chemistryAtty. DocketNo.: 43161-64728 / WO (003WO) tags, barcoding oligos, lysis reagents, permeabilization reagents, fixing reagents, and / or other materials.

[0030] In some embodiments, upon generation, the plurality of partitions is stabilized in position in a close-packed format within a continuous phase, within a region of the collecting container, where each partition has a film or other layer (e.g., a thin film composed of a material that is immiscible with the sample and the continuous phase). As such, the partition matrix can include a set of partitions (e.g., aqueous partitions) generated from a sample, wherein the set of partitions (e.g., aqueous partitions) is stabilized in position in a close- packed format within a continuous phase (e.g., an aqueous continuous phase), and wherein each of the set of aqueous partitions comprises a thin film (or other layer) that is immiscible with the continuous phase (e.g., aqueous continuous phase).

[0031] In some embodiments, generating the plurality of partitions comprises driving a sample fluid through a membrane comprising a distribution of holes, the membrane coupled to a reservoir outlet of a reservoir for the sample fluid, and the reservoir aligned with the collecting container. In some embodiments, the distribution of holes has a density less than 5000 holes per cm2and a hole-to-hole spacing greater than 30 micrometers. In some embodiments, each hole in the distribution of holes has a diameter from 1 through 3 micrometers. In some embodiments, driving the sample fluid through the membrane comprises spinning the sample fluid, the membrane, and the collecting container within a centrifuge in a first direction of rotation, and reversing the direction of rotation, thereby adjusting an equilibrium surface profile of an emulsion comprising the plurality of partitions within the collecting container. In some embodiments, driving the sample fluid through the membrane comprises spinning the sample fluid, the membrane, and the collecting container within a centrifuge at a first rotational v elocity and at a second rotational velocity’ less than the first rotational velocity, thereby adjusting an equilibrium surface profile of an emulsion comprising the plurality of partitions within the collecting container. In some embodiments, the collecting container has a volumetric capacity from 10 through 300 microliters, and wherein each of the plurality of partitions has a characteristic diameter from 10 through 30 micrometers. In some embodiments, the method further comprises transmitting heat to and from the plurality of partitions, within the collecting container, during a heat transmission operation, wherein the temperature varies within a temperature range from 4 °C to 95 °C during the heat transmission operation, and wherein individual partitions of the plurality of partitions remain unmerged with adjacent partitions of the partition matrix in a close-packed format during the heat transmission operation. In some embodiments, generating the pluralityAtty. DocketNo.: 43161-64728 / WO (003WO) of partitions comprises generating greater than 25 million partitions within the collecting container. In some embodiments, generating the plurality of partitions comprises transmitting partitions to form partitions toward a closed end of the collecting container, thereby forming a packed partition matrix at a region of the collecting container away from the membrane, thereby stabilizing the plurality' of partitions in a three dimensional close-packed format (e.g., as a partition matrix) toward the closed end of the collecting container.

[0032] As described in further detail herein, generated partitions of the partition matrix can be fixed and / or immobilized in position using a polymer material. Applications of fixing and / or otherwise immobilizing generated partitions can function to provide calibration samples (e.g., samples where partitions for calibrating interrogation instruments are fixed). Such calibration samples can reduce computational burden associated with calibration of instruments used to interrogate / scan processed samples while running assays, given that no additional algorithmic adjustments would be necessary to run these calibration samples, given that the calibration samples would be more representative of test samples being processed. Applications of fixing and / or otherwise immobilizing generated partitions can function to provide a mechanism by which samples processed remotely can be transported (e.g., shipped) to a test site (e.g., central test site) for sample processing or troubleshooting. Applications of fixing and / or otherwise immobilizing generated partitions can thus also function to increase user accessibility to high performance testing of samples, without requiring users to have an instrument for interrogating / scanning processed samples.

[0033] An additional aspect of the present disclosure provides for a system for generating partitions, the system comprising: a first substrate defining a reservoir comprising a reservoir inlet and a reservoir outlet; a membrane coupled to the reservoir outlet and comprising a distribution of holes; and a second substrate comprising an opening configured to retain a collecting container in alignment with the reservoir outlet, wherein the system comprises: a first operation mode wherein the first substrate is coupled with the second substrate and encloses the collecting container, yvith the reservoir outlet seated within the collecting container, a second operation mode wherein the reservoir contains a sample fluid comprising an aqueous mixture for a digital analysis of target material (e.g., nucleic acid material), and a third operation mode wherein the membrane generates a plurality of partitions that are transmitted into the collecting container to form partitions of the partition matrix at a rate of at least 1 million partitions per minute in response to a force applied to the sample fluid, and a fourth operation mode wherein the plurality of partitions is stabilized in position in a close- packed format, as a partition matrix within a region of the collecting container.Atty. DocketNo.: 43161-64728 / WO (003WO)

[0034] In some embodiments, the membrane is bonded to the reservoir outlet at a perimeter of the reservoir outlet. In some embodiments, the distribution of holes has a density- less than 10,000 holes per cm2and a hole-to-hole spacing greater than 10 micrometers. In some embodiments, the first substrate comprises a set of reservoirs comprising the reservoir; and the system further comprises a set of membranes comprising the membrane, the set of membranes paired with and bonded to outlets of the set of reservoirs; and the second substrate comprises a set of openings comprising the opening, wherein the set of openings is configured to retain a set of collecting containers in alignment with the set of reservoirs. In some embodiments, a reservoir number of the set of reservoirs is different from a collecting container number of the set of collecting containers, wherein the first substrate comprises two or more fluidic pathways from the set of reservoirs to the set of membranes. In some embodiments, the second substrate is configured as a spacer separating the reservoir outlet from a base surface of the collecting container.

[0035] An additional aspect of the present disclosure provides for a method comprising: generating a plurality- of partitions within a collecting container, each of the plurality of partitions comprising an aqueous mixture for a digital analysis of target material (e.g., nucleic acid material), wherein generating the plurality of partitions comprises driving the aqueous mixture through a distribution of holes of a track-etched membrane to be packed toward a closed end of the collecting container, and wherein the plurality of partitions is characterized by less than 13% coefficient of variation for poly dispersity.

[0036] In some embodiments, generating the plurality of partitions includes generating the plurality7of partitions at a rate of at least 600,000 partitions per minute. In some embodiments, the plurality of partitions is characterized by less than 10% occupancy of partition by target nucleic acid material or other target material.

[0037] An additional aspect of the present disclosure provides for a system comprising: a partition matrix within a closed container, wherein a set of partitions of the partition matrix is immobilized within the closed container with a poly mer material, and wherein the partition matrix with the polymer material has a level of optical clarity above a threshold level of clarity. In some embodiments, the set of partitions contains a set and / or distribution of targets. In some embodiments, the set of partitions contains a set and / or distribution of nucleic acid targets. In some embodiments, the set of partitions contains a set and / or distribution of protein targets. In some embodiments, the set of partitions contains at most one target of the set and / or distribution of targets. In some embodiments, the partition matrix with the polymer material has a level of optical clarity without use of refractive indexAtty. DocketNo.: 43161-64728 / WO (003WO) matching between the partition matrix and the polymer material. In some embodiments, the polymer material comprises agarose. In some embodiments, the polymer material comprises a thermoplastic polymer material. In some embodiments, the polymer material comprises a crosslinked polymer material. In some embodiments, each partition of the partition matrix is surrounded by the polymer material. In some embodiments, the polymer material is provided at a surface of the partition matrix as a cap. In some embodiments, the polymer material can be combined with one or more additional components that provide functionality to the polymer, where the additional component(s) can diffuse or otherwise be transmitted into the partitions of the partition matrix (e.g., in order to interact with targets captured in the partitions of the partition matrix). Embodiments of the additional component(s) can include one or more of: chemical probes, affinity ligands, enzymatic components, dyes (e.g.. SYBR Green, hydrolysis probes, non-hydrolysis probes, cell viability-associated dyes, etc.), sequence-specific capture agents (e.g., antisense oligonucleotides, LNA-modified probes, PNA clamps, morpholino oligomers, etc.), CRISPR guide RNAs, molecular beacons, enzymes (e.g., polymerases, reverse transcriptases, ligases, nickases, recombinases, stranddisplacing polymerases, etc.), small molecules (e.g., bis-intercalators (e.g., YOYO-1), minorgroove binders (e.g., netropsin, distamycin), G-quadruplex stabilizers (e.g., TMPyP4, BRACO-19), etc.), cell-penetrant viability' dyes (calcein-AM, SYTO dyes), membrane- impermeant exclusion dyes (7-AAD, TO-PRO-3). lectins, aptamers, antibody-conjugated fluorophores, and metabolic reporters (resazurin, tetrazolium salts), metal-chelating probes, click-chemistry' tags, barcoding oligos, lysis reagents, permeabilization reagents, fixing reagents, and / or other materials.

[0038] An additional aspect of the present disclosure provides methods, systems, devices, and compositions for fixing positions of a set of targets within a matrix in three dimensions. An embodiment of a method can include: fixing positions of a set of targets of a sample within a matrix in three dimensions, wherein the matrix has a level of optical clarity7greater than a threshold level (described in more detail herein), and wherein fixing positions comprises: combining the sample with a set of processing reagents for a reaction, and a gelling material at a temperature above a melting temperature of the gelling material, and reducing the temperature of the sample, the set of processing reagents, and the gelling material below the melting temperature, thereby generating the matrix; linking amplified targets of the set of targets with a set of probes of the set of processing reagents upon performing the reaction; detecting signals emitted from probes of the set of probes associated with targets of the set of targets upon scanning the matrix with an optical detection system; and generating aAtty. DocketNo.: 43161-64728 / WO (003WO) characterization of the set of targets of the sample from the detected signals. In some embodiments, the set of targets comprises a set of nucleic acid targets. In some embodiments, the gelling material comprises at least one of agarose, carrageenan, polyethyleneglycol diacrylate (PEGDA), and polyacrylamide. In some embodiments, the set of processing reagents comprises a set of probes configured to associate with targets of the set of targets upon performing the reaction and to emit fluorescent signals upon associating with targets of the set of targets. In some embodiments, the method further comprises crosslinking the gelling material prior to performing the reaction. In some embodiments, performing the reaction comprises performing the reaction below the melting temperature of the gelling material. In some embodiments, the reaction comprises an isothermal amplification reaction. In some embodiments, the reaction comprises a rolling circle amplification reaction that physically links amplicons of a target template to the target template. In some embodiments, the method further comprises restricting movement of amplicons of a target template away from the target template. In some embodiments, restricting movement comprises coupling amplicons of the target template to a region of the matrix in proximity to the target template. In some embodiments, restricting movement comprises confining amplicons of the target template within a membrane. In some embodiments, generating the characterization comprises one or more of: generating a count of the set of targets; determining co-occurrence of markers associated with each of a set of different analyte types; characterizing changes in detected signals over a set of time points; and characterizing changes in detected signals in response to a stimulus. In some embodiments, the threshold level is at least 80% transmissivity7of light.

[0039] A related embodiment of a method can include: generating a count of a set of targets stabilized in position in three dimensions within a matrix having a level of optical clarity greater than a threshold level, wherein the matrix comprises a gelling material at a temperature below a melting temperature of the gelling material, and wherein generating the count comprises: linking a set of probes with amplicons of the set of targets upon performing a reaction below the melting temperature within the matrix; and detecting fluorescent signals emitted from the set of probes upon scanning a set of cross sections through the matrix with a light sheet system. In some embodiments, the threshold level is at least 80% transmissivity7of light. In some embodiments, the set of targets comprises greater than 1000 targets.

[0040] A related embodiment of a method can include: performing an assay reaction within a matrix in three dimensions with a sample comprising targets (e.g., a set of targets), wherein the method comprises: fixing positions of the targets of the sample in the matrix within aAtty. DocketNo.: 43161-64728 / WO (003WO) reaction vessel (e.g., PCR tube, well of a well plate, other container), wherein fixing positions of the targets comprises: combining the sample with a set of processing reagents for the assay reaction, and a gelling material at a temperature above a melting temperature of the gelling material, and reducing the temperature of the sample, the set of processing reagents, and the gelling material below the melting temperature, thereby generating the matrix; and incubating to allow assay enzymes to generate fluorescent signals upon binding of probes of the set of processing reagents to the targets (e.g., directly to target sequences, indirectly to adapters coupled to target sequences, or complements thereof).

[0041] An embodiment of a system can include: a matrix (e.g., provided within a closed container), the matrix comprising a set of processing reagents for a reaction, and a set of targets stabilized in position in three dimensions within the matrix, wherein the matrix comprises a gelling material at a temperature below a melting temperature of the gelling material, wherein the reaction is configured to be performed below the melting temperature of the gelling material, and wherein the matrix comprises a level of optical clarity' greater than a threshold level. In some embodiments, the set of processing reagents comprises a set of probes configured to associate with amplicons of the set of targets upon performing the reaction and to emit fluorescent signals upon associating with amplicons of the set of targets. In some embodiments, the system further comprises a tight sheet system. In some embodiments, the gelling material comprises at least one of agarose, carrageenan, polyethyleneglycol diacrylate (PEGDA), and polyacrylamide. In some embodiments, the set of targets comprises a set of nucleic acid targets. In some embodiments, the set of targets comprises a set of protein targets. In some embodiments, the set of targets comprises a set of single cells. In some embodiments, the threshold level is at least 80% transmissivity of light.

[0042] An aspect of the disclosure provides embodiments, variations, and examples of a matrix, wherein a set of targets (e.g., single molecules, single analytes, single cells, single nuclei, single proteins, single fragments, single organelles, etc.) is spatially distributed and immobilized within the matrix, in three dimensions. In examples, targets can include one or more of: DNA. RNA, protein, or cells, and / or multianalytes, where various targets can be assayed in parallel. Molecular assays that can be performed within the matrices described could include copy number variation (CNV) assays, single nucleotide profile (SNP) genotyping assays, minimum residual disease (MRD) assays, rare variant detection, gene expression, protein binding or colocalization, or capsid quantitation. Cellular assays could include bacterial pathogen vs host cell quantification, rare cell profiling, immune cell vs cancer cell quantification, live vs. dead cell quantification, or other cellular assays.Atty. DocketNo.: 43161-64728 / WO (003WO)

[0043] In embodiments, the matrix comprises a polymer material and a set of assay components, and the set of targets with the set of assay components and the polymer material has a level of optical clarity above a threshold level of clarity. In some embodiments, the polymer material is a gelling material. The polymer material can include linear polymer structures and / or branched polymer structures. The polymer material can be a thermoplastic or a thermosetting polymer material. In some embodiments, the polymer material comprises a thermoplastic polymer material. In some embodiments, the thermoplastic polymer material comprises agarose. In some embodiments, the polymer material is in a form of a polymer material-containing solution. In some embodiments, the polymer material-containing solution comprises 0.02 to 3% w / v% of agarose. In some embodiments, the polymer material comprises a thermosetting polymer material. The polymer material can be a crosslinked material or a material that is not crosslinked. The matrix can provide or support enzymatic reactions from which signals (e.g., fluorescent signals) associated with the targets dispersed within the matrix can be detected. In examples, detection of signals can be performed using an optical detection system (e.g., light sheet imaging system, other imaging-based optical detection system, other non-imaging-based optical detection system). In vanations, gelling reagents may be heated to about 40 °C, to about 45 °C, to about 50 °C, to about 55 °C, to about 60 °C, to about 65 °C, about 70 °C, to about 75 °C, to about 80 °C, to about 85 °C, to about 90 °C, to about 95 °C, to about 100 °C, to about 105 °C, to about 110 °C, to about 115 °C. to about 120 °C, to about 125 °C, to about 130 °C, to about 135 °C. to about 140 °C. to about 145 °C, to about 150 °C, or greater to dissolve. In variations, gelling reagents may be heated to about 70 °C or greater to dissolve. As such, methods described herein can involve the use of thermostable enzymes. After the matrix sets, the reaction may not be heated above the threshold melting temperature depending upon gelling material / agent used, or the matrix may melt and spatial resolution may be lost. The matrix should not be inhibitory to the assay enzymes and should be sufficiently optically clear to allow for light sheet imaging or detection using other methods. In some embodiments, the matrix is not inhibitory to the assay enzymes and is sufficiently optically clear to allow for light sheet imaging or detection using other methods.

[0044] In comparison with partitioning-based methods (e g., droplet-based partitioning methods, well-based partitioning methods, etc.) for analyzing sample targets, stabilizing positions of targets spatially in a gel matrix would avoid the need to partition targets using other partitioning techniques, which would simplify and improve assay setup times. Such a partition-less technique would also eliminate limitations associated with partition countAtty. DocketNo.: 43161-64728 / WO (003WO) and / or target size thresholds resulting from partition volume limitations (e.g., based on a maximum suitable partition dimension). In variations of using the partition-less methods described herein, the number of targets that could be detected with suitable sensitivity performance would be limited by diffusion in the matrix and the resolution of the imager.

[0045] In variations, the polymer material can be a gelling material. In examples, the gelling material can include at least one of an agarose material, a carrageenan material, a polyethylene glycol (PEG) material, and a polyacrylamide material. In some embodiments, the gelling material comprises an agarose material. In some embodiments, the gelling material comprises an agarose. In some embodiments, the gelling material comprises a carrageenan material. In some embodiments, the gelling material comprises a carrageenan. In some embodiments, the gelling material comprises a polyethylene glycol (PEG) material. In some embodiments, the gelling material comprises a polyethylene glycol (PEG). In some embodiments, the gelling material comprises a polyacrylamide material. In some embodiments, the gelling material comprises a polyacrylamide. Method steps can include setting up an assay reaction with a sample including targets (e.g., a set of targets) of interest, with a gelling material / agent, where the set of targets become fixed within a reaction vessel (e g., PCR tube, well of a well plate, other container) once the gelling material / agent sets. The reaction can then be incubated to allow assay enz mes to generate fluorescent signals upon binding of probes of the reaction to targets of the set of targets (e.g., directly to target sequences, indirectly to adapters coupled to target sequences or complements thereof).

[0046] In examples, assay biochemistry for generation of signals from targets dispersed and / or fixed within the matrix can be based upon polymerase chain reaction (PCR), rolling circle amplification (RCA), isothermal amplification, enzy me-linked immunosorbent assay (ELISA), or another reaction format (e.g., another reaction format as disclosed herein). In examples, detection of signals can be performed using an optical detection system (e.g., light sheet imaging system, other imaging-based optical detection system, other non-imaging- based optical detection system), and positive signals can be counted or otherwise characterized to generate insights regarding aspects of the set of targets of the sample.

[0047] In one specific example, a method can include providing a nucleic acid nanoball including a volume of target nucleic acids; combining the nucleic acid nanoball with a set of processing reagents (e.g., amplification reagents, probes, buffers, etc.) as described in Applications incorporated by reference, where probes of the set of processing reagents do not emit a signal until binding with target nucleic acids (or adapters associated with target nucleic acids) of the volume of target nucleic acids; combining the nucleic acid nanoball and the setAtty. DocketNo.: 43161-64728 / WO (003WO) of processing reagents with a gelling reagent / gelling material (e.g., in a flow state) to form a matrix; allowing the gelling reagent / gelling material to set; performing a reaction within the matrix; and detecting signals associated with the volume of target nucleic acids, from the matrix. In examples, the nucleic acid nanoball can have a characteristic dimension (e.g., diameter) of up to 300 nm, up to 400 nm, up to 500 nm, up to 600 nm, up to 700 nm, up to 800 nm, up to 900 nm, or greater.

[0048] In relation to optical clarity- of the matrix with the set and / or distribution of targets, the threshold level can be a level greater than 60% transmissivity of light, greater than 65% transmissivity7of light, greater than 70% transmissivity7of light, greater than 75% transmissivity- of light, greater than 80% transmissivity of light, greater than 85% transmissivity of light, greater than 90% transmissivity of light, greater than 95% transmissivity of light, greater than 99% transmissivity of light, or greater. In some embodiments, the optical clarity7threshold level can be a level of at least 60% transmissivity of light, at least 65% transmissivity- of light, at least 70% transmissivity of light, at least 75% transmissivity of light, at least 80% transmissivity of light, at least 85% transmissivity of light, at least 90% transmissivity of light, at least 95% transmissivity of light, at least 99% transmissivity of light, or greater. In some embodiments, the optical clarity threshold level is at least 60% transmissivity- of light, at least 70% transmissivity- of light, or at least 80% transmissivity of light. In some embodiments, the optical clarity threshold level is at least 80% transmissivity of light. However, variations of the partition matrix can have optical clarity below 60% transmissivity- of light. In some embodiments, the set and / or distribution of targets with the set and / or distribution of assay components and the polymer material has optical clarity- above the threshold level of clarity without use of refractive index matching between the components of the matrix, and the threshold level is at least 60% transmissivity of light, at least 65% transmissivity of light, at least 70% transmissivity7of light, at least 75% transmissivity of light, at least 80% transmissivity- of light, at least 85% transmissivity of light, at least 90% transmissivity of light, at least 95% transmissivity of light, at least 99% transmissivity of light, or another suitable threshold level. In some embodiments, the set and / or distribution of targets with the set and / or distribution of assay components and the polymer material has optical clarity above the threshold level of clarity without use of refractive index matching between the components of the matrix, and the threshold level is at least 60% transmissivity of light, at least 70% transmissivity- of light, at least 80% transmissivity of light, or another suitable threshold level. In examples, the set and / or distribution of targets with the set and / or distribution of assay components and the polymerAtty. DocketNo.: 43161-64728 / WO (003WO) material has optical clarity above the threshold level of clarity without use of refractive index matching between the components of the matrix, and the threshold level is at least 80% transmissivity of light or another suitable threshold level.

[0049] In embodiments, variations, and examples, the matrix can be a viscous fluid, a shearthickening fluid, a gel (e.g., a gel having individual discrete droplets), or another fluid having a surface. The matrix can be transitioned to a solidified state (e.g., as a solid), where contents distributed within the matrix are immobilized and do not move, even when a force is applied to the matrix and / or the matrix is transported. The matrix can be crosslinked (e.g., in response to a photostimulus, in response to a thermal stimulus, in response to a pH shift, with a chemical crosslinking method, etc ).

[0050] In relation to a single-tube workflow in which the collecting container remains closed (e.g., the collecting container has no outlet, there is no flow out of the collecting container, to avoid sample contamination), method(s) can further include transmitting heat to and from the matrix within the closed collecting container (e.g., according to an assay protocol). In relation to generation of matrices having suitable clarity (e.g., with or without refractive index matching), method(s) can further include transmission of signals from the matrix for readout (e g., by an optical detection platform, by another suitable detection platform).

[0051] Where method(s) include transmitting heat to and from the matrix, the matrix can be configured to be stable across a wide range of temperatures (e.g., 1 °C through 95 °C, greater than 95 °C. greater than 70 °C. less than 1 °C) relevant to various digital analyses and other bioassays, where the positions of targets remain consistent throughout such temperature adjustments.

[0052] The disclosure provides for systems, devices, and methods that enable digital analyses across a wide dynamic range that is 10-100 times greater than that of existing technologies, depending upon application of use. In examples related to nucleic acid counting, the disclosure provides for systems, devices, and methods that can have a dynamic range from 1 through 100 million, due to the high and / or low number of targets that can be distributed within the matrix and distinctly detected upon reading signals associated with the targets.

[0053] In specific applications, systems and methods described herein can perform: detection and counting of nucleic acid molecules via amplification of individual nucleic acid molecule followed by detection of optically detectable signals (e.g., amplification by polymerase chain reaction (PCR) methods, by isothermal methods such as loop-mediated isothermal amplification (LAMP), by recombinase polymerase amplification (RPA), by helicase dependent amplification (HD A), by strand displacement amplification (SDA), by nickingAtty. DocketNo.: 43161-64728 / WO (003WO) enzyme amplification (NEAR), by transcription mediated amplification (TMA), by RNaseH mediated amplification, by whole genome amplification (WGA) using phi29, by rolling circle amplification, etc.) on purified DNA, cDNA, RNA, oligonucleotide tagged antibodies / proteins / small molecules, or directly from lysate (e.g., blood lysate); fluorescent in situ hybridization (FISH) with fluorescently tagged nucleic acids (e.g., PNA, LNA, DNA, RNA, etc.) or an indirect in situ hybridization approach using DIG or biotin, where the signal is later amplified by conjugation of an antibody to alkaline phosphatase or a peroxidase to produce a change in color detected by one or more substrates (e.g., nitroblue tetrazolium (NBT), 5-bromo-4-chloro-3-indolyl-phosphate (BCIP), HNPP, etc.); an in vitro transcription or translation assay whereby a colorimetric or fluorescent reporter is used for detection; droplet PCR applied to samples derived from single cells (e.g., prokaryotes, eukaryotes), organelles, viral particles, and exosomes; enumeration of protein or peptide molecules (e.g., by proximity ligation assays, etc.); sequencing applications (e.g., single molecule sequencing applications); monitoring or detection of products (e.g., proteins, chemicals) released from single cells (e.g., interleukin released from immune cells); monitoring cell survival and / or division for single cells; monitoring or detection of enzymatic reactions involving single cells; antibiotic resistance screening for single bacteria; enumeration of pathogens in a sample (e.g., in relation to infections, sepsis, in relation to environmental and food samples, etc.); enumeration of heterogeneous cell populations in a sample; enumeration of individual cells or viral particles (e.g., by encapsulating cells in droplets with species-specific antibodies coupled with enzymes that react with substrate components in the droplet to produce signals, etc.); monitoring of viral infections of a single host cell; liquid biopsies and companion diagnostics; prenatal diagnosis of genetic disorders (e g., aneuploidy, genetically inherited diseases) such as with cell-free nucleic acids, fetal cells, or samples containing mixtures of fetal and maternal cells; detection of cancer forms from various biological samples (e.g., detection of cancer from cell-free nucleic acids, tissue biopsies, biological fluids, feces); detection of markers (e.g., genetic markers) relevant to the field of oncology; detection and / or monitoring of minimal residual diseases; monitoring responses to therapies; detection or prediction of rejection events of transplanted organs; linkage analysis; detection of titer characteristics (e.g., viral titer characteristics); detection of integrity; detection of full vs. empty capsid markers; other diagnostics associated with other health conditions; other characterizations of statuses of other organisms; and other suitable applications.Atty. DocketNo.: 43161-64728 / WO (003WO)

[0054] In specific applications, the systems, devices, and methods can perform processes that achieve goals of digital PCR, quantitative PCR, and next generation sequencing, using a single platform.

[0055] In embodiments, the target material analyzed according to digital analysis and / or other bioassay techniques can include one or more of: nucleic acid material (e.g., DNA, RNA, miRNA, etc.), protein material, amino acid material, other small molecules, other single analytes, other multi-analytes, and / or other suitable target material of a sample. In embodiments, the sample can include or otherwise be derived from: whole tissue structures, tissue portions (e.g., histological tissue slices, formalin-fixed paraffin-embedded (FFPE) tissue, frozen tissue, biopsied tissues, fresh frozen plasma, seeded natural scaffolds, seeded synthetic scaffolds, etc.), organs, whole organisms, organoids, cell suspensions (e.g.. frozen cell suspensions that are separated prior to processing with the system, cell suspensions retained in a medium / hydrogel medium, etc.), nuclei suspensions, other suspensions, single cells, organelles, sub-organelle structures, intra-organelle components, viruses, microorganisms, and other samples.

[0056] Another aspect of the present disclosure provides a non-transitory computer readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein.

[0057] Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.

[0058] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. The present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.4. INCORPORATION BY REFERENCE

[0059] All publications, patents, and patent applications mentioned in this specification, including U.S. Provisional Application No. 63 / 725,548 and 63 / 830,353, are herein incorporated by reference in their entireties for all purposes and to the same extent as if eachAtty. DocketNo.: 43161-64728 / WO (003WO) individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.5. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also '‘figure” and ‘"FIG.” herein), of which:

[0061] FIG. 1 depicts an embodiment of a system for generating partitions.

[0062] FIG. 2 depicts a first example of a system for generating partitions.

[0063] FIG. 3 depicts a variation of a fastener configuration in a system for generating partitions.

[0064] FIG. 4 depicts a variation of a fastener configuration in a system for generating partitions.

[0065] FIG. 5 depicts an example of a method for assembling components of a system for generating partitions.

[0066] FIG. 6 depicts a variation of a configuration of a system for generating partitions.

[0067] FIG. 7A depicts a second example of a system for generating partitions.

[0068] FIG. 7B depicts a third example of a system for generating partitions.

[0069] FIG. 7C depicts examples of collecting containers of a system for generating partitions.

[0070] FIG. 8A depicts a flow chart of an embodiment of a method for generating partitions.

[0071] FIG. 8B depicts a flow chart of an embodiment of a method for immobilizing partitions of a partition matrix.

[0072] FIGs. 9A-9C depict examples of methods for producing desired surface characteristics of partitions generated according to methods described herein.

[0073] FIG. 9D depicts an example of the driving solution, the sample fluid, the continuous aqueous phase, and the immiscible layer, within a units of an example of the sy stem described herein.

[0074] FIG. 10 depicts a flow chart of an embodiment of a method for generating partitions.Atty. DocketNo.: 43161-64728 / WO (003WO)

[0075] FIG. 11A depicts an embodiment of a system for fixing and detecting targets.

[0076] FIG. 11B depicts examples of components of a system for fixing and detecting targets.

[0077] FIG. 12A depicts a flow chart of an embodiment of a method for fixing and detecting targets.

[0078] FIG. 12B depicts a flow chart of a variation of a method for fixing and detecting targets.

[0079] FIG. 13 illustrates a computer system that is programmed or otherwise configured to implement methods provided herein.6. DETAILED DESCRIPTION OF THE INVENTION(S)

[0080] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions can occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be employed.

[0081] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2. or 3 is equivalent to greater than or equal to 1 , greater than or equal to 2, or greater than or equal to 3.

[0082] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0083] Furthermore, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that 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 specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.Atty. DocketNo.: 43161-64728 / WO (003WO)6.1. General Overview

[0084] The present disclosure covers systems, devices, methods performed by such systems and devices, and methods of manufacturing and assembling such devices. Generally, embodiments of the device include assemblies of reservoirs, functionalized membranes, and supporting bodies for collecting containers, where the assemblies rapidly produce large numbers of monodisperse partitions from a sample that can be assayed in a manner that achieves goals of digital PCR, quantitative PCR, and next generation sequencing in a high- performance manner, and with a single platform. Partitions produced by such devices are stabilized in a three dimensional format within closed collecting containers, thereby providing a "single-tube" workflow that eliminates risk of sample cross-contamination from initial reception of a sample, to distributing of the sample across a high number of partitions, to performance of reactions within individual partitions, to detecting signals generated by contents of individual partitions from with the closed collecting containers.

[0085] Partition matrices described herein can be immobilized / ixed in position using polymer materials, as described herein, without compromising partition matrix clarity, such that the partition matrix can still be interrogated for detection of signals emitted from partition contents (e.g., from within the collecting container used to hold generated partitions).

[0086] In some embodiments, the polymer material can be combined with one or more additional components that provide functionality to the polymer, where the additional component(s) can diffuse or otherwise be transmitted into the partitions of the partition matrix (e.g., in order to interact with targets captured in the partitions of the partition matrix). Embodiments of the additional component(s) can include one or more of: chemical probes, affinity ligands, enzymatic components, dyes (e.g., SYBR Green, hydrolysis probes, nonhydrolysis probes, cell viability-associated dyes, etc.), sequence-specific capture agents (e.g., antisense oligonucleotides, LNA-modified probes, PNA clamps, morpholino oligomers, etc.), CRISPR guide RNAs, molecular beacons, enzymes (e.g., polymerases, reverse transcriptases, ligases, nickases, recombinases, strand-displacing polymerases, etc ), small molecules (e.g., bis-intercalators (e.g., YOYO-1), minor-groove binders (e.g., netropsin, distamycin), G- quadruplex stabilizers (e g., TMPyP4, BRACO-19), etc ), cell-penetrant viability dyes (calcein-AM, SYTO dyes), membrane-impermeant exclusion dyes (7-AAD, TO-PRO-3), lectins, aptamers, antibody-conjugated fluorophores, and metabolic reporters (resazurin, tetrazolium salts), metal-chelating probes, click-chemistry tags, barcoding oligos, lysis reagents, permeabilization reagents, fixing reagents, and / or other materials.Atty. DocketNo.: 43161-64728 / WO (003WO)

[0087] The systems, methods, and devices disclosed herein can provide several additional benefits over other systems and methods, and such systems, methods, and devices are further implemented into many practical applications across various disciplines.

[0088] Partition matrices generated according to methods described herein, using systems described herein, can be fixed and / or immobilized such that they can be transported (e.g., shipped) prior to being analyzed using an instrument (e.g., light sheet scanning instrument). As such, a partition matrix generated as described herein can be dropped, thrown, impacted, or subject to mechanical forces without disrupting positions of individual partitions (i.e., a set of partitions of the partition matrix). In embodiments where such partition matrices are scanned using multiple channels (e.g., excitation channels, emission channels) of an instrument used for interrogation, fixing and / or immobilizing partitions in such a manner allows for multiplexed analyses of partition contents using the multiple channels available.

[0089] In relation to optical clarity, immobilized or otherwise positionally-stabilized partitions can be clear (e.g., allowing greater than 60% transmissivity of light, greater than 65% transmissivity of light, greater than 70% transmissivity’ of light, greater than 75% transmissivity of light, greater than 80% transmissivity of light, greater than 85% transmissivity of light, greater than 90% transmissivity of light, greater than 95% transmissivity of light, greater than 99% transmissivity of light, or greater). In relation to thermal characteristics, immobilized or otherwise positionally-stabilized partitions can be subjected to heating and / or cooling, as appropriate for various biological assays or other processes.

[0090] In relation to fixing and / or immobilization of partitions, devices, methods, and systems of the present disclosure can provide a streamlined workflow where polymer materials used for fixation and / or immobilization of partitions are transmitted over the partitions in a flow state (e.g., a continuous flow state) at a first temperature, and cool (e.g., during centrifugation) to a second temperature to arrive at a set state, thereby immobilizing the partitions in a single step workflow. In some embodiments, the first temperature is a glass transition temperature of the polymer material, a melting temperature of the polymer material, or any other suitable temperature. In some embodiments, the second temperature is a temperature below the glass transition temperature of the polymer material, a temperature below the melting temperature of the polymer material, or any other suitable temperature. In some embodiments, the first temperature is at least 40 °C. at least 45 °C, at least 50 °C, at least 55 °C, at least 60 °C, at least 65 °C, at least 70 °C. at least 75 °C, at least 80 °C, at least 85 °C, at least 90 °C, at least 95 °C, at least 100 °C, at least 105 °C, at least 110 °C, at leastAtty. DocketNo.: 43161-64728 / WO (003WO)115 °C, at least 120 °C, at least 125 °C, at least 130 °C, at least 135 °C, at least 140 °C, at least 145 °C, at least 150 °C. any other intermediate value, or greater. In some embodiments, the first temperature is 40 °C or lower, 45 °C or lower, 50 °C or lower, 55 °C or lower, 60 °C or lower, 65 °C or lower, 70 °C or lower, 75 °C or lower, 80 °C or lower, 85 °C or lower, 90 °C or lower, 95 °C or lower, 100 °C or lower, 105 °C or lower, 110 °C or lower, 115 °C or lower, 120 °C or lower, 125 °C or lower, 130 °C or lower, 135 °C or lower, 140 °C or lower, 145 °C or lower, 150 °C or lower, any other intermediate value, or lower.

[0091] Polymer materials used can include linear polymer structures and / or branched polymer structures. The polymer material can be a thermoplastic or a thermosetting polymer material. In some embodiments, the polymer material comprises a thermoplastic polymer material. In some embodiments, the thermoplastic polymer material comprises agarose. In some embodiments, the polymer material is in a form of a polymer material-containing solution. In some embodiments, the polymer material -containing solution comprises 0.02 to 3% w / v% of agarose. In some embodiments, the polymer material comprises a thermosetting polymer material. The polymer material can be a crosslinked material or a material that is not crosslinked.

[0092] Devices, methods, and systems of the present disclosure may generate a plurality of partitions at a high rate, where the partitions are stabilized in position (e.g., in a close-packed format, in equilibrium stationary positions) within a collecting container. Notably, the partitions are stable across a wide range of temperatures (e.g., 1 °C through 95 °C, greater than 95 °C, less than 1 °C) relevant to various digital analyses and other bioassays, where the partitions remain consistent in morphology7and remain unmerged with adjacent partitions. As discussed, stabilization of the partitions within a continuous phase is further performed in a manner where the emulsion has a high degree of clarity (e.g., at least 50% transmissivity of light, at least 60% transmissivity of light, at least 70% transmissivity of light, at least 80% transmissivity of light, at least 90% transmissivity of light, at least 99% transmissivity of light, etc.), such that signals from cross-sections of the emulsion within the collecting container can be interrogated (e.g., using a 3D imaging technique, using a planar imaging technique, etc.). Such clarity characteristics persist even after immobilization with the polymer material, without requiring or involving refractive index matching between components of the partition matrices (e.g., sample, immiscible phase, continuous phase, polymer material).

[0093] The devices, systems, and methods disclosed herein can further generate partitions in a consistent and controlled manner (e.g., as monodisperse, uniform partitions with a lowAtty. DocketNo.: 43161-64728 / WO (003WO) degree of poly dispersity) for applications in biotechnology' (e.g., with respect to microscale and nanoscale assays) or other fields.

[0094] The devices, systems, and methods disclosed herein further provide a alternative to partition generation using microfluidic devices, in a manner that generates partitions in a consistent and reliable manner. In specific examples, the systems and devices may include assemblies with track-etched membranes for producing partitions from a sample provided within a reservoir, in a consistent and controlled manner (e.g., as monodisperse / uniform partitions). In these examples, hole density and / or hole-to-hole spacing of the membranes is significantly lower than that which may be used for membrane-based filtration.

[0095] The present disclosure also provides disposable devices and methods for generating partitions using a single-tube and closed tube workflow from partition generation through reaction performance within individual partitions, through optical interrogation of partition contents, thereby preventing sample cross contamination throughout digital analyses processes.

[0096] Additionally or alternatively, in variations, the devices, methods, and systems disclosed herein can be adapted for transmitting a high number of partitions, where the partitions are generated at a high rate, to various well formats and tube formats, thereby improving performance of existing droplet-based systems.

[0097] In examples, the systems, methods, and devices of the present disclosure have applications in digital amplification of nucleic acid molecules (e.g.. digital polymerase chain reaction (PCR), digital loop-mediated isothermal amplification (LAMP), digital multiple displacement amplification (MDA), digital recombinase polymerase amplification (RPA), digital helicase dependent amplification, reverse transcription, in vitro transcription and translation, overlap extension amplification, etc ).

[0098] In examples, the systems, methods, and devices of the present disclosure have applications in single cell and organelle capture (e.g., for mammalian cells, for bacterial cells, for pathogens, for viral particles, for organelles, etc.).

[0099] In examples, the systems, methods, and devices of the present disclosure have applications in single, double, or other emulsion generation. For instance, the systems, methods, and devices disclosed herein can use one or more of centrifugation, pressure, or other forces to disperse a fluid, as partitions, through one or more layers of fluids (i.e., ‘continuous fluids') that are immiscible with each other, forming emulsions.

[0100] In examples, the systems, methods, and devices of the present disclosure can have applications in microparticle generation and liposome generation for other applications.Atty. DocketNo.: 43161-64728 / WO (003WO)

[0101] The present disclosure also covers related systems, devices, and methods performed by such systems and devices. Generally, embodiments of the device support stable spatial sets and / or distributions of sample targets, where the sample targets can be assayed in a manner that achieves goals of digital PCR, quantitative PCR, and next generation sequencing in a high-performance manner, and with a single platform. Targets processed using such devices are stabilized in a three-dimensional format within a matrix (e.g.. optically clear matrix) within a closed collecting container, thereby providing a ‘"single-tube” workflow that eliminates risk of sample cross-contamination from initial reception of a sample, to performance of reactions within the matrix, to detecting signals generated by contents of the matrix from with the closed collecting containers.

[0102] Matrices described herein can be immobilized / fixed in position using gelling materials, as described herein, without compromising matrix clarity, such that the matrix can still be interrogated for detection of signals emitted from the matrix contents (e.g., from within the collecting container used to hold the matrix).

[0103] The systems, methods, and devices disclosed herein can provide several additional benefits over other systems and methods, and such systems, methods, and devices are further implemented into many practical applications across various disciplines.

[0104] Matrices generated according to methods described herein, using systems described herein, can fix and / or immobilize targets such that they can be transported (e g., shipped) prior to being analyzed using an instrument (e.g., light sheet scanning instrument and / or system). As such, a matrix generated as described herein can be dropped, thrown, impacted, or subject to mechanical forces without disrupting positions of targets distributed within the matrix). In embodiments where such matrices are scanned using multiple channels (e.g., excitation channels, emission channels) of an instrument used for interrogation, fixing and / or immobilizing targets in such a manner allows for multiplexed analyses of matrix contents using the multiple channels available.

[0105] In relation to optical clarity, immobilized or otherwise positionally-stabilized targets can be distributed across a matrix that is clear (e.g., allowing greater than 60% transmissivity of light, greater than 65% transmissivity of light, greater than 70% transmissivity of light, greater than 75% transmissivity of light, greater than 80% transmissivity of light, greater than 85% transmissivity' of light, greater than 90% transmissivity of light, greater than 95% transmissivity of light, greater than 99% transmissivity of light, or greater). In relation to thermal characteristics, immobilized or otherwise positionally-stabilized targets of the matrixAtty. DocketNo.: 43161-64728 / WO (003WO) can be subjected to heating and / or cooling, as appropriate for various biological assays or other processes.

[0106] In relation to fixing and / or immobilization of targets, devices, methods, and systems of the present disclosure can provide a streamlined workflow where polymer materials (e.g., gelling materials) used for immobilization of targets are combined with a sample and processing reagents in a flow state (e.g., a continuous flow state) at a first temperature (e.g., a temperature above a melting temperature, a temperature above a glass transition temperature), and cool to a second temperature (e.g., a temperature below the melting temperature, , a temperature below a glass transition temperature) to arrive at a set state, thereby immobilizing the targets in a single step workflow. Polymer and / or gelling materials used can include linear polymer structures and / or branched polymer structures. The polymer and / or gelling material can be a thermoplastic or a thermosetting material. In some embodiments, the polymer and / or gelling material comprises a thermoplastic material. In some embodiments, the thermoplastic material comprises agarose. In some embodiments, the polymer and / or gelling material is in a form of a polymer and / or gelling material-containing solution. In some embodiments, the polymer and / or gelling material-containing solution comprises 0.02 to 3% w / v% of agarose. In some embodiments, the polymer and / or gelling material comprises a thermosetting material. The polymer and / or gelling material can be a crosslinked material or a material that is not crosslinked.

[0107] The devices, systems, and methods disclosed herein further provide a alternative to approaches involving partitions (e.g., using droplets, using microfluidic devices, etc.), in a manner that spatially distributes targets for observation, in a consistent and reliable manner.

[0108] In examples, the systems, methods, and devices of the present disclosure have applications in digital amplification of nucleic acid molecules (e.g.. digital polymerase chain reaction (PCR), digital loop-mediated isothermal amplification (LAMP), digital multiple displacement amplification (MDA), digital recombinase polymerase amplification (RPA), digital helicase dependent amplification, reverse transcription, in vitro transcription and translation, overlap extension amplification, etc ).

[0109] In examples, the systems, methods, and devices of the present disclosure have applications in single cell and organelle capture (e.g., for mammalian cells, for bacterial cells, for pathogens, for viral particles, for organelles, etc.).

[0110] Additionally or alternatively, the systems, devices, or methods described herein can confer any other suitable benefit.Atty. DocketNo.: 43161-64728 / WO (003WO)6.2. Systems

[0111] As shown in FIG. 1, an embodiment of a system 100 for generation of partitions includes: a first substrate 1 10 defining a set of reservoirs 114, each having a reservoir inlet 115 and a reservoir outlet 116; one or more membranes 120 positioned adjacent to reserv oir outlets of the set of reservoirs 114, each of the one or more membranes 120 including a distribution of holes 125; and optionally, a sealing body 130 positioned adjacent to the one or more membranes 120 and including a set of openings 135 aligned with the set of reservoirs 114; and optionally, one or more fasteners (including fastener 140 shown in FIG. 1) configured to retain the first substrate 110, the one or more membranes 120, and optional the sealing body 130 in position relative to a set of collecting containers 150. In variations, the system 100 can additionally include a second substrate 160. wherein the one or more membranes 120 and optionally, the sealing body 130, are retained in position between the first substrate 110 and the second substrate 160 by the one or more fasteners.

[0112] Embodiments of the system 100 function to generate a plurality7of partitions at a high rate (e.g., of at least 50.000 partitions / minute, of at least 100,000 partitions / minute of at least 200,000 partitions / minute, of at least 300,000 partitions / minute, of at least 400,000 partitions / minute, of at least 500,000 partitions / minute, of at least 600,000 partitions / minute, of at least 700,000 partitions / minute, of at least 800,000 partitions / minute, of at least 900,000 partitions / minute, of at least 1 million partitions / minute, of at least 2 million partitions / minute, of at least 3 million partitions / minute, etc.), where the partitions are stabilized in position (e.g., in a close-packed format, in equilibrium stationary positions) within a collecting container. Rates of partitions generation can be average rates determined in relation to duration of applied force.

[0113] Embodiments of the system 100 function to fix and / or immobilize partition matrices that are generated, by enabling processing of generated partition matrices with polymer materials that can positionally fix and / or immobilize partitions within the collecting container.

[0114] Embodiments of the system may further function to reliably generate partitions in a consistent and controlled manner (e.g., as monodisperse and uniform partitions having little- to-no poly dispersity) for various applications, such as digital amplification and analysis and other assays; capture of target material at cellular, subcellular, and molecular scales; sample analyses benefitting from partition generation; or other suitable applications, with high- performance achievement of goals of digital PCR, quantitative PCR, next generation sequencing, and other assays for analysis of samples. Embodiments of the system 100 alsoAtty. DocketNo.: 43161-64728 / WO (003WO) function to generate partitions using devices that are non-microfluidic, disposable or reusable, in a cost-effective manner.

[0115] Embodiments of the system 100 can be used to implement one or more steps of methods disclosed herein. However, the system 100 can additionally or alternatively be configured to perform other suitable methods.

[0116] An embodiment of a system can include: a partition matrix within a closed container, wherein a set of partitions of the partition matrix is immobilized within the closed container with a polymer material, and wherein the partition matrix with the polymer material has a level of optical clarity above a threshold level of clarity. In some embodiments, the set of partitions contains a set and / or distribution of targets. In some embodiments, the set of partitions contains a set and / or distribution of nucleic acid targets. In some embodiments, the set of partitions contains a set and / or distribution of protein targets. In some embodiments, the set of partitions contains at most one target of the set and / or distribution of targets. In some embodiments, the partition matrix with the polymer material has a level of optical clarity without use of refractive index matching between the partition matrix and the polymer material. In some embodiments, the polymer material comprises agarose. In some embodiments, the polymer material comprises a thermoplastic polymer material. In some embodiments, the polymer material comprises a crosslinked polymer material. In some embodiments, each partition of the partition matrix is surrounded by the polymer material. In some embodiments, the polymer material is provided at a surface of the partition matrix as a cap. In some embodiments, the polymer material can be combined with one or more additional components that provide functionality to the polymer, where the additional component(s) can diffuse or otherwise be transmitted into the partitions of the partition matrix (e.g., in order to interact with targets captured in the partitions of the partition matrix). Embodiments of the additional component(s) can include one or more of: chemical probes, affinity ligands, enzy matic components, dyes (e.g., SYBR Green, hydrolysis probes, nonhydrolysis probes, cell viability-associated dyes, etc.), sequence-specific capture agents (e.g., antisense oligonucleotides, LNA-modified probes, PNA clamps, morpholino oligomers, etc.), CRISPR guide RNAs, molecular beacons, enzymes (e.g., polymerases, reverse transcriptases, ligases, nickases, recombinases, strand-displacing polymerases, etc ), small molecules (e.g., bis-intercalators (e.g., YOYO-1), minor-groove binders (e.g., netropsin, distamycin), G- quadruplex stabilizers (e g., TMPyP4, BRACO-19), etc ), cell-penetrant viability dyes (calcein-AM, SYTO dyes), membrane-impermeant exclusion dyes (7-AAD, TO-PRO-3), lectins, aptamers, antibody-conjugated fluorophores, and metabolic reporters (resazurin,Atty. DocketNo.: 43161-64728 / WO (003WO) tetrazolium salts), metal-chelating probes, click-chemistry tags, barcoding oligos, lysis reagents, permeabilization reagents, fixing reagents, and / or other materials.

[0117] As show n in FIG. 11 A, an embodiment of a system 400 for fixing positions of a set of targets of a sample within a matrix in three dimensions includes: a matrix 410 (e.g., provided within a closed container 420), the matrix 410 comprising a set of processing reagents for a reaction, and a set of targets stabilized in position (e.g.. random positions, nonrandom positions, pre-selected positions, etc.) in three dimensions within the matrix. In embodiments, the matrix includes a gelling material at a temperature below a melting temperature of the gelling material, the reaction is configured to be performed below the melting temperature of the gelling material, and the matrix comprises a level of optical clarity greater than a threshold level. In some embodiments, the system further comprises a light sheet system for scanning cross sections of the matrix 410 and / or other samples.

[0118] In relation to optical clarity, the threshold level can be characterized as allowing greater than 60% transmissivity7of light, greater than 65% transmissivity of light, greater than 70% transmissivity of light, greater than 75% transmissivity7of light, greater than 80% transmissivity of light, greater than 85% transmissivity of light, greater than 90% transmissivity of light, greater than 95% transmissivity of light, greater than 99% transmissivity7of light, or greater. In some embodiments, the optical clarity7threshold level can be characterized as allowing at least 60% transmissivity of light, at least 65% transmissivity of light, at least 70% transmissivity of light, at least 75% transmissivity of light, at least 80% transmissivity of light, at least 85% transmissivity of light, at least 90% transmissivity7of light, at least 95% transmissivity7of light, at least 99% transmissivity7of light, or greater. In some embodiments, the optical clarity threshold level can be characterized as allowing at least 60% transmissivity7of light, at least 70% transmissivity of light, or at least 80% transmissivity7of light. In some embodiments, the optical clarity threshold level can be characterized as allowing at least 80% transmissivity of light.

[0119] In embodiments, variations, and examples, the set and / or distribution of targets can include one or more of: nucleic acid targets, protein targets, viral particles, exosomes, cells (e.g., single cells), cell parts, and / or any combination of various target types. In some embodiments, the set and / or distribution of targets comprises a set and / or distribution of nucleic acid targets. In some embodiments, the set and / or distribution of nucleic acid targets comprises a set and / or distribution of nucleic acid targets encoding a set and / or distribution of protein targets. In some embodiments, the set and / or distribution of targets comprises a set and / or distribution of protein targets. In some embodiments, the set and / or distribution ofAtty. DocketNo.: 43161-64728 / WO (003WO) targets comprises a set and / or distribution of single cells. The set and / or distribution of targets can be suspended within and / or immobilized within the matrix during scanning using optical detection systems described herein, where aspects of the matrix are described in more detail herein.

[0120] In embodiments, variations, and examples, the set of processing reagents can include a set of probes configured to associate with targets and / or amplicons of the set of targets upon performing the reaction and to emit fluorescent signals upon associating with targets and / or amplicons of the set of targets. As such, the targets can be directly or indirectly fluorescently- labeled by the set of probes. The set of probes can include sequences configured to interact with target sequences, sequences complementary to target sequences, and / or adapter sequences associated with target sequences (e.g., where adapters can be added during amplification reactions). The set of probes can include dyes that emit a signal when associated with target sequences, and the signals can be detected with amplification of target sequences. In some embodiments, association and / or interaction of the set of probes of the set of processing reagents with targets and / or amplicons of the set of targets comprises hybridization (e.g., based on complementary sequences). In some embodiments, each probe of the set of probes is configured to emit a fluorescent signal.

[0121] In examples, dyes (e.g., for tagging of RNAs, DNAs, oligonucleotides, etc.) can include one or more of: FAM, (e.g., 6-FAM), Cy3TM, Cy5TM, Cy5.5TM, TAMRATM (e.g., 5-TAMRA, 6-TAMRA, etc ), MAX, JOE. TETTM. ROX, TYETM (e.g., TYE 563, TYE 665, TYE 705, etc.), Yakima Yellow ®, HEX, TEX (e.g., TEX 15), SUN, ATTOTM (e.g., ATTO 488, ATTO 490LS, ATTO 532, ATTO 550, ATTO 565, ATTO RhoIOl, ATTO 590, ATTO 633, ATTO 647, ATTO 647N, etc.), Alexa Fluor ® (e.g., Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 594, Alexa Fluor 647. Alexa Fluor 660. Alexa Fluor 750, etc.), IRDyes® (e.g., 5’IRDye 700, 5’IRDye 800, 5TRDye 800CW, etc.). Rhodamine (e.g., Rhodamine Green, Rhodamine Red, Texas Red ®, Lightcycler ®, Dy 482XL, Dy 508XL, Dy 526XL, Dy 750, Hoechst dyes, DAPI dyes, SYTOX dyes, chromomycin dyes, mithramycin dyes, YOYO dyes, ethidium bromide dyes, acridine orange dyes, TOTO dytes, thiazole dyzes, CyTRAK dyes, propidium iodide dyes, LDS dyes, BODIPY dyes, and / or other dyes. In some embodiments of the systems, methods, devices, and compositions disclosed herein, the dyes used are selected from: Alexa Fluor 488, Alexa Fluor 594, ATTO 490LS, ATTO 532, ATTO 647N, Cy5TM, FAM, Dy 482XL, Dy 508XL, Dy 526XL, and combinations thereof. In examples, cell function dyes for tagging of target material and detection can include one or more of: DCFH, DHR, SNARF, indo-1, Fluo-3, Fluo-4, and / orAtty. DocketNo.: 43161-64728 / WO (003WO) other dyes. In examples, fluorescent proteins for tagging of target material and detection can include one or more of: cerulean, mCFP, mTurquoise, T-Sapphire, CyPet, ECFP, CFP. EBFP, Azurite, and / or other fluorescent proteins.

[0122] In an embodiment, dyes used for 10-plex or greater multiplexing of targets using the matrix 410 or other sample formats can include: Alexa Fluor™ 488, Atto™ 532, Alexa Fluor™ 594, Atto™ 647N, Cy5. FAM, Dy-526XL, Dy-508XL, Dy-482XL, and Atto™ 490LS.

[0123] As such, detection of labeled targets can involve fluorescent nucleic acid probes, protein markers, lipid markers, carbohydrates markers, or other optically-detectable markers.

[0124] Other aspects of the set of processing reagents include embodiments, variations, and examples of processing reagents described in U.S. Application No. 18 / 085.217 filed on 20- DEC-2022, which is herein incorporated in its entirety by this reference.

[0125] In embodiments, variations, and examples, the matrix can be gel-like, where examples of a gel-like matrix include a hy drogel matrix. Material of the matrix can be provided in a liquid state (e.g.. at a temperature above the melting temperature), a solid state (e.g., at a temperature below the melting temperature), or a transition state between a liquid state and a solid state. Hydrogels can be stimulus-responsive (e g., temperature responsive, pH responsive electric field responsive, light-responsive, etc.). While upper critical solution temperature (UCST) hydrogels are described, the hydrogels can alternatively be lower critical solution temperature (LCST) hydrogels that transition from a liquid to a solid as the temperature increases (e.g., as in a pluronic material). The matrix may comprise a hydrogel material. In some embodiments, the matrix comprises a hydrogel material. In some embodiments, the matrix does not comprise a hydrogel material.

[0126] In embodiments, variations, and examples of hydrogel matrices, the gelling material of the matrix 410 can include at least one of agarose, gelatin, carrageenan, polyacrylamide, polyethyleneglycol (PEG), polyethyleneglycol diacrylate (PEGDA), gellan gum, agar, or other suitable gelling material. In some embodiments, the gelling material of the matrix includes agarose. In some embodiments, the gelling material of the matrix includes gelatin. In some embodiments, the gelling material of the matrix includes carrageenan. In some embodiments, the gelling material of the matrix includes polyacrylamide. In some embodiments, the gelling material of the matrix includes polyethyleneglycol (PEG). In some embodiments, the gelling material of the matrix includes polyethyleneglycol diacrylate (PEGDA). In some embodiments, the gelling material of the matrix includes gellan gum. In some embodiments, the gelling material of the matrix includes agar. However, other gellingAtty. DocketNo.: 43161-64728 / WO (003WO) materials that have suitable melting temperature properties, optical clarity characteristics, and characteristics that do not result in inhibiting reaction performance, can be used.

[0127] In some embodiments, the melting temperature of the gelling material is at least 40 °C, at least 45 °C, at least 50 °C, at least 55 °C, at least 60 °C, at least 65 °C, at least 70 °C, at least 75 °C, at least 80 °C, at least 85 °C, at least 90 °C, at least 95 °C, at least 100 °C, at least 105 °C, at least 110 °C, at least 115 °C, at least 120 °C, at least 125 °C. at least 130 °C, at least 135 °C, at least 140 °C. at least 145 °C, at least 150 °C, any other intermediate value, or greater. In some embodiments, the melting temperature of the gelling material is 40 °C or lower, 45 °C or lower, 50 °C or lower, 55 °C or lower, 60 °C or lower, 65 °C or low er, 70 °C or lower, 75 °C or lower, 80 °C or lower, 85 °C or lower, 90 °C or lower, 95 °C or lower, 100 °C or lower, 105 °C or lower, 110 °C or lower, 115 °C or lower, 120 °C or lower, 125 °C or low er, 130 °C or low er, 135 °C or lower. 140 °C or lower, 145 °C or lower, 150 °C or lower, any other intermediate value, or low er.

[0128] In practice, generating the matrix can include mixing a sample comprising targets and processing reagents with material of the matrix in a precursor form (e.g., liquid form at a first temperature), and then transitioning the matenal of the matrix to a second form (e.g., solid form at a second temperature, upon crosslinking, etc.). After distributing the set of targets of the sample throughout the matrix, the matrix can be crosslinked (e.g., photocrosslinked, chemically crosslinked, crosslinked with a pH shift, crosslinked with a temperature shift, physically crosslinked, or crosslinked in another suitable manner). The matrix may be crosslinked. In some embodiments, the matrix is crosslinked. In some embodiments, the matrix is not crosslinked.

[0129] In an alternative embodiment, each target unit (e.g., molecule, cell, nucleus, other unit) can be isolated in a sub-matrix component (e.g., gel. bead, etc.) and then aggregated together (e.g., container), followed by adding a gelling material or reagent to form a bulk matrix.

[0130] In an alternative embodiment, the set of targets can be stabilized in position within a fluid, such as a density gradient suspending solution that reduces or eliminates mobility of the set of targets (e.g., during scanning or optical interrogation).

[0131] In embodiments, the matrix 410 can be optically clarified upon generation (e g., during combination with the set of targets and / or the set of processing reagents).Alternatively, the matrix 410 can be optically clarified at some point after generation, such as after the set of targets and / or the set of processing reagents are dispersed throughout the matrix in solid form, after target amplification, etc. Clarification can involve the use of aAtty. DocketNo.: 43161-64728 / WO (003WO) refractive index-matching fluid, a clarifying agent that can be applied to the matrix or used to surround the collecting container 420, or another suitable agent.

[0132] The set of targets can be freely suspended in the matrix 410 or fluid (e.g., fluid with a densify gradient, as described herein). In relation to distribution of the set of targets in a matrix 410, the set of targets can be anchored in the matrix 410 by way of crosslinking (e.g. for nucleic acids or other molecules, template molecules can be modified to have a functional group, such as an acrydite group, that crosslinks into hydrogels during polymerization). In a specific example, target nucleic acid molecules can be modified to have an acrydite group that crosslinks with PEGDA during polymerization.

[0133] The matrix 410 can be configured to restrict movement of amplicons of a target template (e.g., template target nucleic acid) away from the target template. Amplicons can thus be physically linked to the target template (e.g. using rolling circle amplification of a circular DNA template, etc.). As such, a reaction within the matrix can include a rolling circle amplification reaction that physically links amplicons of a target template to the target template. Restricting movement can additionally or alternatively include coupling amplicons of the target template to a region of the matrix in proximity to the target template. Restricting movement can additionally or alternatively include physically linking amplicons to the matrix as they are formed (e.g. using primers of the set of processing reagents that have anchors that crosslink with matrix 410). Restricting movement can additionally or alternatively include confining amplicons of the target template within a membrane. For instance, amplicons of a target template can be confined to within a cell or viral particle due to their biological membranes or cellular structures. In a specific example, gene targets of a suspension of fixed cells be amplified and retained in position within fixed cell membranes (e.g. with performance of a FISH-like reaction in fixed cells to detect if cells carry a mutation or gene target). The amplified targets within membranes can be suspended or immobilized, as described herein, and then optically interrogated. Target amplicons can additionally or alternatively have restricted diffusion aw ay from an originating target template due to the network structure of the matrix 410.

[0134] Embodiments of the system 400 can be used to implement one or more steps of methods disclosed herein. For instance, upon detection of signals from the set and / or distribution of targets suspended or immobilized in a matrix, various analyses characterizing the set of targets can be performed. Generating a characterization of the set and / or distribution of targets can include one or more of: generating a count of the set and / or distribution of targets (e.g., targets labeled by fluorescent markers); determining coAtty. DocketNo.: 43161-64728 / WO (003WO) occurrence of markers associated with each of a set of different analyte types (e.g., molecular types, cellular types, etc.); characterizing changes in detected signals over a set of time points (e.g., in relation to monitoring growth of a culture, in relation to monitoring amplification of signals over the span of a reaction, etc.); and characterizing changes in detected signals in response to a stimulus (e.g., environmental factor, heat stimulus, for melt curve analysis, an electromagnetic stimulus [e.g.. gene expression resulting from exposure to an electromagnetic stimulus, uncaging of reaction components by UV light, such as with caged polymerase or dNTPs], a chemical stimulus [e.g., with infusion of chemicals into the matrix that affects signal intensities, with use of grow th factors for cells or tissues, with use of a denaturant for DNA or proteins], etc.).

[0135] Ho ever, the system 400 can additionally or alternatively be configured to perform other suitable methods.6.2.1. Systems - Supporting Substrates / Housing6.2.1.1. Supporting Substrates - First Substrate

[0136] FIG. 1 depicts an embodiment of a system 100, where the system 100 includes a first substrate 110 defining a set of reservoirs 114, each having a reservoir inlet 115 and a reservoir outlet 116. The first substrate 110 can function to provide at least a portion of a coupling mechanism for retaining positions of other elements of the system 100 relative to one or more collecting containers 150 described in more detail herein. The first substrate 110 also functions to receive and stage fluid to be transmitted to the membrane layer 120 for formation of partitions on the opposing side of the membrane layer, where the partitions pass into contents of the collecting container to form a partition matrix. The first substrate 110 can be configured to disposable (e.g., composed of inexpensive, recyclable, and / or compostable materials), such that the system 100 can provide a alternative for generation of partitions. Additionally or alternatively, the first substrate 110 can be configured to be a reusable element (e.g., usable for generation of partitions in multiple runs of the system 100).

[0137] In order to provide a robust mechanism of coupling with other system elements (e.g., supporting bodies for the collecting containers 150, a second substrate 160 described herein, etc.), the first substrate 1 10 can be composed of a material having suitable mechanical properties. In variations, materials of the first substrate 110 can be configured to provide suitable mechanical properties in relation to stresses attributed to flow' through set of reservoirs 114 (e.g., stresses due to centrifugation, stresses due to pressurization, radial stresses, shear stresses, longitudinal stresses, tensile stresses, compressive stresses, stressesAtty. DocketNo.: 43161-64728 / WO (003WO) associated with impacts to the system 100 during use; stresses due to thermal expansion, stresses due to thermal contraction, and other associated stresses depending upon applications of use).

[0138] Additionally or alternatively, the first substrate 110 can be composed of a material having suitable thermal properties. In variations, materials of the first substrate 110 can be configured to provide suitable thermal properties in relation to one or more of: thermal conductivity (e.g., in relation to heating or cooling of fluids from which partitions are generated by the system 100) and other thermal properties depending upon application of use.

[0139] Additionally or alternatively, the first substrate 110 can be composed of a material having suitable physical or surface properties. In variations, materials of the housing 121 and / or other aspects of the interface 120 can be configured to provide suitable physical or surface properties in relation to one or more of: non-reactiveness with sample materials; low porosity (e.g., so as to not absorb sample material); high hydrophobicity; and other suitable phy sical or surface properties.

[0140] Additionally or alternatively, the first substrate 110 can be composed of a material having suitable optical properties (e.g., for optical interrogation of sample materials, for protecting sample materials from electromagnetic radiation, etc.). In variations, the first substrate 110 is translucent, transparent, or otherwise has a high degree of transparency, to support optical interrogation of sample materials or sample processing materials within the set of reservoir(s) 114. Furthermore, translucent or transparent characteristics of the first substrate 1 10 may enable operation modes in which contents of the set of reservoirs 1 14 can be visualized (e.g., during manual filling, during automatic filling), such that a level of material within each reservoir can be verified prior to generation of partitions using the system 100. As such, the first substrate 110 can be composed of a material having a suitable level of transparency. Still alternatively, the first substrate 1 10 can be composed of an opaque material.

[0141] In variations, the first substrate 110 can be composted of a synthetic material or a natural material. In examples, the first substrate 110 can be composed of a polymeric material (e.g., a polyetheretherketone, an acetal, an acrylonitrile butadiene styrene, a nylon, a polycarbonate, a polypropylene, a polystyrene, etc.); a metallic material, a ceramic material, a composite material, or another suitable material. The first substrate 110 can be fabricated by machining, printing (e.g., 3D printing), molding (e.g., injection molding), or through another suitable method.Atty. DocketNo.: 43161-64728 / WO (003WO)

[0142] In variations, the first substrate 110 can have a broad surface at which access (e.g., through openings) into the set of reservoirs 114 is provided. As shown in FIG. 1. each reservoir can have a reservoir inlet 1 15 and a reservoir outlet 116. The reservoir inlet 115 can be defined as an open surface at the broad surface of the first substrate 110, and the reservoir outlet can be defined as an open surface at a second surface of the first substrate 110. Each reservoir can define a cavity for receiving fluid from which partitions are generated. In examples the cavity can have a volume from 1 pL through 100 mL; however, variations of the cavity can have another suitable volume. The cavity can be constant in cross section (e g., transverse cross section) from the reservoir inlet 115 to the reservoir outlet 116.Alternatively, the cavity may not be constant in cross section from the reservoir inlet 115 to the reservoir outlet 116. For instance, a portion of the cavity near the reservoir inlet 115 can include morphology (e.g., flared morphology) configured to complement a fluid deli v ery device (e.g., a pipette tip), thereby facilitating fluid transfer to the system 100 for partition generation. Additionally or alternatively, the cavity can define a non-straight or non-linear path from the reservoir inlet 115 to the reservoir outlet 116. Furthermore, in variations, a reservoir of the set of reservoirs 114 can have a reservoir inlet 115 at any other suitable surface of the first substrate 110, a reservoir outlet 116 at any other suitable outlet of the first substrate 110, or define another suitable fluid path from the reserv oir inlet 115 to the reservoir outlet 116.

[0143] One or more filters can further be positioned within the set of reservoirs 114 and / or otherwise positioned upstream of the one or more membranes 120 described in further detail herein, where the filter(s) can function to remove undesired material and prevent undesired material from entering partitions or disrupting partition formation. In examples, the filter(s) can be structured to allow passage of targets (e.g.. nucleic acids, proteins, cells, viral material, chemicals, analytes, etc.) from the sample(s) for distribution across the plurality of partitions, where such filter(s) can accordingly have suitable sizes, porosity, hydrophobicity', surface charge, and / or other characteristics.

[0144] The example of the first substrate 110a shown in FIG. 2 includes four reservoirs corresponding to four collecting containers (e.g., of a complementary apparatus) for generated partitions; however, the first substrate 110 can alternatively define another suitable number of reservoirs (e.g., from one to 10,000 reservoirs). Furthermore, the number of reservoirs may not correspond to the number of collecting containers in a one-to-one manner. For instance, the first substrate 110 can define a number of reservoir inlets fewer than that of the number of collecting containers, where the reservoirs can be configured to branch alongAtty. DocketNo.: 43161-64728 / WO (003WO) their respective lengths and terminate at outlets corresponding to the collecting containers. As such, a number of samples can be distributed across a number of collecting containers, where the number of collecting containers is greater than the number of samples. Alternatively, the first substrate 110 can define a number of reservoir inlets greater than that of the number of collecting containers. As such, contents of multiple reservoirs can be combined prior to distribution across a set of collecting containers (e.g., in variations in which samples and processing materials are mixed within reservoirs prior to generation of partitions). As such, a reservoir number of the set of reservoirs can be different from a collecting container number of the set of collecting containers, and the first substrate can include one or more fluidic pathways (or two or more fluidic pathways) from the set of reservoirs to the set of membranes.

[0145] The first substrate 110 can include features configured to receive or position the set of fasteners 140 (described in more detail herein) for coupling with other elements of the system 100, in order to enable retention of the elements of the system 100 in position relative to each other. Such features maintain alignment and relative positioning between system elements and provide proper sealing of elements (e.g., membranes to reservoir outlets). In the example shown in FIG. 2, the set of fasteners 140a can include a set of protrusions at the first substrate 110a, that complement tabs at the second substrate 160a.

[0146] Alternatively, as shown in FIG. 3, the first substrate 110c can include a set of flanges defining through-holes through the flanges with an orientation parallel to the broad surface of the first substrate 1 10b, such that the set of fasteners including fastener 140b pass parallel to the broad surface through corresponding openings of a complementary element (e.g., second substrate 110b, collecting container supporting body, etc.). In this variation, separation of the first substrate 110 from the complementary element would apply shear to the set of fasteners.

[0147] Still alternatively, the first substrate 1 10 may not define any openings or through holes, but enable fastening using another suitable mechanism. For instance, the first substrate 110 can include one or more of: protrusions (e.g., tabs, as shown in FIG. 4) that form a portion of a snap-fit mechanism between the first substrate 110c and recesses of a complementary element (e.g., second substrate 160c, collecting container supporting body, etc.); magnetic elements that enable magnetic coupling with a complementary element (e.g., second substrate 110, collecting container supporting body, etc.); adhesive elements that couple with a complementary element (e.g., second substrate 110, collecting container supporting body, etc.); and other suitable coupling mechanisms. The configurations shown inAtty. DocketNo.: 43161-64728 / WO (003WO)FIGs. 2, 3, and 4 provide reliable coupling between elements of the system 100, without providing obstacles to accessing the reservoir(s) of the first substrate.

[0148] Additionally or alternatively, the first substrate 110 can include features configured to receive or position the set of fasteners 140 for coupling to other elements of the system 100, in order to enable retention of the elements of the system 100 in position relative to each other. In one variation, the first substrate 110 can include a set of through holes passing perpendicular to the broad surface of the first substrate 110, in order to enable fastening between the first substrate 110 and a complementary element (e.g., second substrate 110, collecting container supporting body, etc.) with the membrane layer 120 and optionally, the sealing body 130 positioned (e.g., sandwiched, compressed) between the first substrate 110 and the complementary element. In this variation, separation of the first substrate 110 from the complementary element would apply tension to the set of fasteners 150.6.2.I.2. Supporting Substrates - Second Substrate

[0149] In variations, the system 100 can additionally include a second substrate 160, wherein the one or more membranes 120 and optionally, the sealing body 130 are retained in position between the first substrate 110 and the second substrate 160 by the one or more fasteners 140 described in more detail herein. In variations, the second substrate 160 can define openings (including second opening 60 shown in FIG. 1) corresponding to the set of reservoirs 114 and / or set of collecting containers 150. such that partitions formed upon transmission of fluid through the one or more membranes 120 pass through openings of the second substrate 160 and into the set of collecting containers 150. The openings of the second substrate 160 can further retain the collecting containers in position and / or in alignment with respective reservoir outlets, such that generated partitions are transferred directly into the collecting containers.

[0150] An example of the second substrate 160 is shown in FIG. 2, where the set of fasteners, including fastener 140a, couples the first substrate 110a to the second substrate 160, with the membranes 120a retained in position between the first substrate 110 and the second substrate 160. The second substrate 160a can be identical to or different from the first substrate 110 in material composition and / or properties described herein. Furthermore, the second substrate 160a can include or define channels through which fluid passes into the collecting containers in a desired manner.

[0151] In the example shown in FIG. 2, the reservoir 114a is partially formed by the first substrate 110a and a reservoir channel 14a, where the reservoir channel 14a and the openingAtty. DocketNo.: 43161-64728 / WO (003WO) of the first substrate 110a cooperate to define a volume into which a material (e.g., sample fluid, process fluid, etc.), can be received for partitioning. As shown in FIG. 2, the first substrate 110a and the reservoir channel 14a can be sealed against each other (e.g., by a seal 130a, by using compliant materials, such that biasing the first substrate 110a against the reservoir channel 14a forms a seal, etc.). Furthermore, as shown in FIG. 2, the membrane 120a can be positioned at an outlet of the reservoir channel 14a and seated within the second substrate 160a, or pass through the second substrate into a respective collecting container 150a. In variations, the membrane 120a can be bonded to the reservoir outlet of the reservoir channel 14a, or retained in position in another manner (e.g., by being seated between the reservoir channel 14a and the second substrate 160a).

[0152] While embodiments, variations, and examples of the first substrate 110 described hereininclude descriptions of a set of reservoirs, variations of the first substrate 1 10 and / or second substrate 160 can alternatively define a single reservoir or pathway into a single collecting container.6.2.2. Systems - Membrane

[0153] As shown in FIG. 1, the system 100 may include one or more membranes 120 positioned adjacent to reservoir outlets of the set of reservoirs 114. The membrane layer 120 includes a distribution of holes 125, through which fluid from the set of reservoirs 114 passes or is driven to generate partitions, which pass into contents of the collecting container to form partition matrices for various applications. The one or more membranes 120 can further function to provide a alternative to microfluidic devices for generation of partitions, where the one or more membranes 120, along with other elements of the system 100 described herein, can generate partitions of a desired morphology (e.g., target partition size in relation to sample volume and target total number of partitions) and composition in a consistent (e.g., with high uniformity, with low merging, with low- poly dispersity) and reliable manner, and at an extremely rapid rate.

[0154] In relation to generation of partitions in a controlled manner, a membrane 120 of the system 100 can include or be composed of one or more track-etched membranes (e.g., membrane sheets) with precisely defined hole sizes, membrane thicknesses and hole densities. For generation of partitions, fluid in the one or more reservoirs upstream of the membrane layer 120 is forced through the distribution of holes 125 by way of applied forces (e.g., centrifugation, pressurized gas, etc.). The partitions formed upon exit of the distribution of holes 125 are then transmitted from the membrane 120 into one or more respectiveAtty. DocketNo.: 43161-64728 / WO (003WO) collecting containers described in more detail herein to form partition matrices, where the collecting containers contain a fluid (e.g., air, a fluid that is immiscible with the partition material, etc.) and / or layers of fluids to control partition composition and morphology. While the membrane 120 can be track-etched to generate the distribution of holes in a precise manner (e.g., with respect to size, shape, and density ), the membrane 120 can alternatively be generated without ion track techniques. For instance, the membrane 120 can alternatively be generated using another method (e.g., laser etching, chemical etching, electroporation, etc.).

[0155] As shown in FIG. 1, the one or more membranes 120 are configured to be retained in position adjacent to the reservoir outlets 116 of the first substrate 110. In variations, the membrane(s) 120 can thus be positioned within the reservoirs of the set of reservoirs 114 described herein. Alternatively, the membrane layer 120 can be positioned outside of the reservoir outlets (e.g., at an external terminal portion of the reservoir 114, downstream of the reservoir outlets 116) of the first substrate 110.

[0156] In variations, the membrane(s) 120 can include a continuum of material positioned across all of the one or more reservoir outlets of the set of reservoirs, for instance, by sandwiching or otherwise retaining the membrane(s) 120 in position downstream of the reservoir outlets of the first substrate 110. Alternatively, the one or more membranes 120 can include separate bodies or regions of material positioned at terminal regions or within reservoirs of the first substrate 110. In variations wherein the one or more membranes 120 include separate bodies or regions of material, the separate bodies / regions can each have different characteristics (e.g., with respect to reservoirs carrying different types of fluids, with respect to generation of different size partitions, etc.). As such, in some variations, each reservoir can have its own associated membrane region that is separate from adjacent membrane regions. In variations where the one or more membranes 120 are divided, individual membrane regions can be divided by a physical barrier (e.g., a gap, a body of non- porous / non-absorbent material, etc.), or divided by another suitable barrier.

[0157] The one or more membranes 120 can be coupled to other adjacent system elements (e.g., the first substrate 110, reservoir outlets 116, optional sealing body 130, the second substrate 160, etc.) by one or more of: an adhesive, a thermal bond, mechanical bond, chemical bond, laser welding, ultrasonic welding, a light-cured adhesive, a temperature-cured adhesive, a moisture-cured adhesive, injection-molding (e.g., as a single piece using overmolding), and / or another suitable bonding mechanism.

[0158] FIG. 5 depicts an example method of bonding a membrane 120d to a reservoir outlet 16d of a reservoir 116d, at the perimeter of the reservoir outlet 116d. As shown in FIG. 5,Atty. DocketNo.: 43161-64728 / WO (003WO) light-curable (e.g., ultraviolet light-curable) adhesive 12d is applied to the perimeter of an exterior portion of the reservoir outlet 16d. Then, material of the membrane (e.g., track- etched polycarbonate, track-etched polyethylene, etc.) is applied to the reservoir outlet 16d at the perimeter, followed by application of a release film 17d over the material of the membrane 120d. The light-curable adhesive 12d is then exposed to light of appropriate wavelength(s) (e.g., ultraviolet light), followed by removal of the release film 17d and excess material of the membrane 120d. In variations, however, the membrane 120 can be coupled to the first substrate 110 and / or the reservoir 116 in another suitable manner.

[0159] In variations, the one or more membranes 120 can be composed of a polymeric material (e.g., polycarbonate, polyester, polyimide). In a first specific example, the one or more membranes 120 are polycarbonate track-etched (PCTE) membranes. In a second specific example, the one or more membranes 120 are polyethylene track-etched membranes. However, the one or more membranes 120 can alternatively be composed of another suitable material processed in another suitable manner.

[0160] In variations, the distribution of holes 120 can be generated in bulk membrane material with specified hole diameter(s), hole depth(s) (e.g., in relation to membrane thickness), aspect ratio(s), hole density, and hole orientation, where, in combination with fluid parameters, the structure of the membrane can achieve desired flow rate characteristics, with reduced or eliminated poly dispersity and merging, and steady formation of partitions (e.g.. without jetting of fluid from holes of the membrane).

[0161] In variations, the hole diameter can range from 0.2 micrometers to 30 micrometers, and in examples, the holes can have an average hole diameter can be 0.02 micrometers, 0.04 micrometers, 0.06 micrometers, 0.08 micrometers, 0.1 micrometers, 0.5 micrometers, 1 micrometers. 2 micrometers. 3 micrometers, 4 micrometers, 5 micrometers, 6 micrometers, 7 micrometers, 8 micrometers, 9 micrometers, 10 micrometers, 20 micrometers, 30 micrometers, any intermediate value, or greater than 30 micrometers (e.g., with use of membrane having a thickness greater than or otherwise contributing to a hole depth greater than 100 micrometers).

[0162] In variations, the hole depth can range from 1 micrometer to 200 micrometers (e.g., in relation to thickness of the membrane layer) or greater, and in examples the hole depth (e.g., as governed by membrane thickness) can be 1 micrometers, 5 micrometers, 10 micrometers, 20 micrometers, 30 micrometers, 40 micrometers, 50 micrometers, 60 micrometers. 70 micrometers, 80 micrometers, 90 micrometers, 100 micrometers, 125Atty. DocketNo.: 43161-64728 / WO (003WO) micrometers, 150 micrometers, 175 micrometers, 200 micrometers, or any intermediate value.

[0163] In variations, the hole aspect ratio can range from 5: 1 to 200: 1, and in examples, the hole aspect ratio can be 5: 1, 10: 1, 20: 1, 30: 1, 40: 1, 50: 1, 60: 1, 70: 1, 80: 1, 90: 1, 100: 1, 125: 1, 150: 1, 175:1, 200: 1, or any intermediate value.

[0164] In variations, the hole density can range from 100 holes / cm2to 15000 holes / cm2(e.g., in order to prevent partitions generated from neighboring holes from merging together as they exit the holes, in relation to spin time, speed, and / or pressurization, etc.). In examples, the hole density can be 100 holes / cm2, 200 holes / cm2, 300 holes / cm2, 400 holes / cm2, 500 holes / cm2, 600 holes / cm2, 700 holes / cm2, 800 holes / cm2, 900 holes / cm2, 1000 holes / cm2, 2000 holes / cm2, 3000 holes / cm2, 4000 holes / cm2, 5000 holes / cm2, 6000 holes / cm2, 7000 holes / cm2, 8000 holes / cm2, 9000 holes / cm2, 10,000 holes / cm2, 11,000 holes / cm2, 12,000 holes / cm2, 13,000 holes / cm2, 14,000 holes / cm2, 15,000 holes / cm2, or any intermediate value. In a specific example, the density of holes is less than 10,000 holes / cm2.

[0165] In variations, the hole-to-hole spacing can range from 5 micrometers to 200 micrometers or greater, and in examples, the hole-to-hole spacing is 5 micrometers, 10 micrometers, 20 micrometers, 30 micrometers, 40 micrometers, 50 micrometers, 60 micrometers, 70 micrometers, 80 micrometers, 90 micrometers, 100 micrometers, 125 micrometers, 150 micrometers, 175 micrometers, 200 micrometers, or greater. In a specific example, the hole-to-hole spacing is greater than 10 micrometers.

[0166] In variations, the membrane 120 can have a diameter or other characteristic dimension from 1 to 50 micrometers, and in examples, the diameter or other characteristic dimension can be 1 micrometer, 2 micrometers, 3 micrometers, 4 micrometers, 5 micrometers. 6 micrometers. 7 micrometers, 8 micrometers, 9 micrometers, 10 micrometers. 20 micrometers, 30 micrometers, 40 micrometers, 50 micrometers, or any intermediate value.

[0167] In examples, the hole orientation can be substantially vertical (e.g., during use in relation to a predominant gravitational force), otherwise aligned with a direction of applied force through the distribution of holes, or at another suitable angle relative to a reference plane of the membrane layer 120.

[0168] In specific examples, the thickness of the membrane layer can be from 23-125 micrometers, with a hole density of less than 10,000 holes / cm2and a hole diameter from 1 to 3 micrometers for generation of water-in-oil-in water (WOW) partitions approximately 14-30 micrometers in diameter under gravitational force of 16000g without observation of partition merging during formation. In the specific examples, partitions were generated form a sampleAtty. DocketNo.: 43161-64728 / WO (003WO) volume of 50 microliters, having a fluid density of 1255 kg / m3, a fluid viscosity of 0.007 Ns / m2, and a surface tension of 0.07 N / m. One specific example of the membrane 120 was characterized by a membrane thickness of 125 micrometers, a hole diameter of 1.5 micrometers, a hole density of 5000 / cm2, an average partition diameter of 30 micrometers, a poly dispersity of -12.1 (CV, %), under a duration of centrifugation at 16,000g for 10 minutes.

[0169] However, other fluid compositions and characteristics can be used, such as those described in U.S. Pat. No. 11,162,136 granted on 02-NOV-2021, which is herein incorporated in its entirety by this reference.

[0170] In relation to surface properties, the membrane layer 120 can be treated or coated with a hydrophobic material in order to provide improved consistency of partitions (e.g.. in relation to consistent partition sizes, in relation to controlled partition sizes, in relation to monodispersity’, etc.). The hydrophobic material can additionally function to improve heat stability of partitions. In variations, the hydrophobic material can include one or more of: an oil, a polysiloxane (e.g., hydroxy-terminated poly dimethylsiloxane), a fluorocarbon-coated silica with a polymer binder; a perfluoroalkyl methacrylate copolymer with our without a distribution of substrates (e.g., nanoparticle substrates); a polystyrene material (e.g., manganese oxide polystyrene, zinc oxide polystyrene), precipitated calcium carbonate, carbon nanotubes, fluorinated silanes, fluoropolymer coatings, silica-based coatings, nanocoatings. and / or other suitable hydrophobic or superhydrophobic materials. The material coating can be processed or otherwise selected to produce desired characteristics in relation to contact angle characteristics (e.g., static contact angle, contact angle hysteresis), sliding angle characteristics, and / or other suitable characteristics.

[0171] In variations, the hydrophobic / superhydrophobic material can be applied to the membrane layer 120 by one or more of: dip coating, spray coating, deposition (e.g., chemical vapor deposition), in-situ growth, polymerization, plasma coating, and / or another suitable coating technique.

[0172] Additionally or alternatively, the membrane layer 120 can include or be treated with other components or coatings to provide desired functions in relation to one or more of: electrostatic charge shielding (e.g., to prevent adsorption of proteins and other biomolecules, to improve partition stability ), protection of the membrane layer (e.g., from degradation), and / or another suitable function.

[0173] Desired partition sizes can be produced based upon a set of factors associated with the membrane layer 120 and applied forces. Parameters of the membrane layer 120 can beAtty. DocketNo.: 43161-64728 / WO (003WO) improved for generation of monodisperse partitions for generation of partition matrices or other uses in relation to any one or more of Weber number; other factors in relation to fluid inertia, surface tension, or other factors. In more detail with respect to Weber number, the membrane layer 120 can be configured with a suitable characteristic hole dimension D (e.g., hole depth, hole diameter), intended fluid density p, governing fluid velocity v during partition generation, and fluid surface tension o. where Weber number We = py^D a, relating drag forces to cohesion forces in relation to partition generation from the membrane layer. In examples, We significantly less than 1 produces periodic dripping for generation of monodisperse partitions, We ~ equal to 1 produces chaotic dripping for generation of poly disperse partitions, and We greater than 1 produces jetting without partition generation. In relation to membrane parameters described herein, increasing hole depth / thickness of the membrane 120 from 50 micrometers to 125 micrometers decreases the We by approximately 6.5 fold, and adjusting the hole diameter from 1 to 3 micrometers changes We from 5.7e-6 to 1.395e-3.

[0174] Relatedly, parameters associated with the membrane layer 120, fluid characteristics, and applied force characteristics can be improved for generation of partitions having a desired size. In a variation for partition formation with an air gap between the membrane layer 120 and a collection fluid within the collecting container, partition radius R is a function of hole radius rc, surface tension of the aqueous phase yaq, fluid density of the partition fluid pw, and acceleration force G (e.g., associated with applied centrifugation forces, associated with pressurization, etc.), where R ~ [[rc / a9] / [2tyvG]]1 / 3.

[0175] In a variation for partition formation without an air gap between the membrane layer 120 and a collection fluid (e.g., an oil) within the collecting container, partition radius R is a function of hole radius rc, surface tension of the interface yinterfaciai, fluid density of the partition fluid paq, fluid density of the collection fluid pon. and acceleration force G (e.g., associated wdth applied centrifugation forces, associated with pressurization, etc.), where R ~

[0176] In specific examples, the resultant partition size can be from 10 micrometers to 80 micrometers; however, variations of the membrane layer 120 and / or system 100, as well as applied force, fluid densities of the fluid to be partitioned and the receiving fluid, interfacial tension of the fluid to be partitioned, can be configured to generate partition with any other suitable dimensions.Atty. DocketNo.: 43161-64728 / WO (003WO)6.2.3. Systems - Seals

[0177] As shown in FIG. 1, the system 100 can optionally include one or more sealing bodies 130 positioned adjacent to the membrane layer 120 and including a set of openings 135 aligned with the set of reservoirs 114. The sealing body 130 functions to promote fluid transmission in a desired manner from the reservoir(s) of the first substrate 110 and through the membrane layer 120 to the collecting container(s), without leakage from the system 100 in an undesired manner. However, in some variations, the system 100 can omit a sealing body, such as in variations where the membrane layer 120 is bonded to other system elements directly, for instance, via light-curable bonding, heat bonding, or laser welding (e.g., an example of which is shown in FIGs. 7A-7B and described herein in relation to FIG. 5).

[0178] In variations, the sealing body 130 can include a first portion upstream of the membrane layer 120. Additionally or alternatively, the sealing body 130 can include a second portion downstream of the membrane layer. In such variations, the portion(s) of sealing body 130 can be compressed or otherwise retained against the membrane layer (e.g., by way of the set of fasteners 150, the first substrate 110, and / or the second substrate 160). In these variations, the sealing body 130 portions can include or otherwise define openings aligned with and corresponding to the set of reservoirs 114 or the set of collecting containers described in more detail herein. Additionally or alternatively, in a variation shown in FIG. 6, the sealing body 130 can additionally or alternatively include a portion (e.g., gasket or o-ring) positioned within a reservoir of the first substrate 110, in order to seal against a fluid delivery or pressurization device configured to drive fluid to or through the membrane layer 120 for partition formation.

[0179] In variations, the sealing bodies 130 can include one or more of: an adhesive (e.g. optically-cured adhesive), heat-bonded seal, sealing element (e.g., o-rings, gasket, etc.), sealing film or paste, or other sealing element.6.2.4. Systems - Fastener(s) and Collecting Container(s)

[0180] As shown in FIG. 1, the system 100 can include one or more fasteners 140 configured to retain the first substrate 110, the membrane layer 120, and the sealing body 130 in position relative to a set of collecting containers 150. The one or more fasteners 140 function to compress or otherwise retain elements of the system 100 in position properly, with respect to forces applied to the system during partition formation.

[0181] In variations, the fasteners 140 can include one or more of: screws, pins, plungers, protrusions (e.g., tabs), recesses (e.g., recesses configured to mate with protrusions),Atty. DocketNo.: 43161-64728 / WO (003WO) magnetic elements, adhesives, bonded couplers (e.g., thermally bonded couplers), and / or other suitable fasteners. Variations of fasteners are shown in FIGs. 2. 3, and 4 as described herein, wherein in the variation shown in FIG. 4, the fasteners 140 include protrusions that form a portion of a snap-fit mechanism with recesses of a complementary element (e.g., second substrate 110, collecting container supporting body, etc.).

[0182] As shown in FIGs. 1, 2, 6, and 7A-7C, the system 100 can be configured to complement, mate with, or otherwise interface with one or more collecting containers 150 for receiving generated partitions. FIG. 7A depicts an example of the system 100, with a set of reservoirs, including reservoir 114e defined within first substrate 1 lOe, where a set of membranes, including membrane 120e, is bonded to reservoir outlets of the set of reservoirs (e.g., using the process shown and described in relation to FIG. 5). The system shown in FIG. 7A further includes second substrate 160e, which supports and aligns collecting containers, including collecting container 150e, with the set of reservoirs, such that the set of membranes is positioned within the collecting containers during use of the system. During use, the first substrate 1 lOe is thus assembled with the second substrate 160e and collecting containers, which is positioned within supporting body 170e for application of a force to generate partitions.

[0183] In variations, the system 100 can be configured in a manner such that there is a gap (e.g., for air, for another fluid) between the system 100 and the collecting container(s). In such variations, one of which is shown in FIG. 7B, the second substrate can be configured as a spacer 160f separating the membrane 120f (at the respective reservoir outlet) from a base surface 5 Of of the collecting container 15 Of. As such, during partition generation with one or more fluid layers within the collecting container 15 Of, the membrane 120f can be spaced above a fluid layer (FIG. 7B, top right), such that partitions generated from the membrane pass through air or other fluids prior to hitting the fluid layer. However, the substrate 160 may not be configured as a spacer, as shown in FIG. 7B (bottom right), thereby positioning the reserv oir outlet 116e directly within a fluid layer (e.g., non-aqueous phase, oil phase, for an aqueous sample fluid) for partition generation. As such, the system 100 can be configured in a manner such that there is no gap between the system 100 and the collecting container(s) and generated partitions are transmitted directly from the system into liquid layers within respective collecting containers.

[0184] In variations, the collecting container(s) can include any suitable number of containers with desired volumetric capacities. In examples (some of which are shown in FIGs. 7A-7C), the collecting container(s) 150 can be configured with one or more of theAtty. DocketNo.: 43161-64728 / WO (003WO) following formats: 0.2ml tube format, strip tubes (e.g., 8x strip tube format), microtiter plate format (e.g., 96-well plate format, 48-well plate format. 24-well plate format, 12-well plate format, etc.), 1.5ml tube format, conical tube format (e.g., 15ml conical format, 50ml conical format, etc.), or another suitable format. The collecting container(s) can be disposable or reusable. In some variations, the collecting container(s) can be supported by a supporting body 170 configured to position the system 100 properly with respect to force-applying apparatus. As shown in FIGs. 1 and 2, the collecting containers can be supported by a supporting body 170, 170a, respectively, which can function as a swing bucket for positioning of the system 100 within a centrifuge for force application, in order to generate partitions. However, the supporting body can be configured in another suitable manner.

[0185] The system 100 can, however, include other suitable elements for generation of partitions in a desired manner. Furthermore, the system 100 can be configured to transition between various operation modes, including: a first operation mode wherein the first substrate is coupled with the second substrate and encloses the collecting container, with the reservoir outlet seated within the collecting container (an example of which is shown in FIG. 7A), a second operation mode wherein the reservoir contains a sample fluid (an embodiment of which is shown in FIG. 1), and a third operation mode w herein the membrane generates a plurality7of partitions that pass into contents to form partition matrices within the collecting container at a high rate (e.g., of at least 1 million partitions per minute, at other rates described herein) in response to a force applied to the sample fluid, and a fourth operation mode wherein the plurality of partitions is stabilized in position in a close-packed format within a region of the collecting container. In relation to the operation modes described herein, structural configurations of the system or contents of the system can produce partitions with a low degree of polydispersity (e.g., less than 15% coefficient of variation for poly dispersity, less than 14% coefficient of variation for polydispersity, less than 13% coefficient of variation for polydispersity, less than 12% coefficient of variation for polydispersity, less than 11% coefficient of variation for polydispersity, less than 10% coefficient of variation for polydispersity, less than 9% coefficient of variation for polydispersity, less than 8% coefficient of variation for polydispersity, less than 7% coefficient of variation for polydispersity, less than 6% coefficient of variation for polydispersity, less than 5% coefficient of variation for polydispersity, etc.) at an unprecedented rate, for digital analyses and other applications, as described herein.

[0186] As such, generating the plurality of partitions can include driving the aqueous mixture through a distribution of holes of a membrane (e.g., track-etched membrane) toAtty. DocketNo.: 43161-64728 / WO (003WO) stabilized positions toward a closed end of the collecting container, the plurality of partitions having significantly low poly dispersity, as described herein.

[0187] Aspects of methods performed by embodiments, variations, and examples of the system 100 are described in more detail herein.

[0188] As shown in FIG. 1 IB, the sy stem 400 can include one or more collecting containers 420 for containing the matrix with the set and / or distribution of targets, for supporting performance of the reaction(s) (e.g., reactions that amplify and label targets of the set of targets), and for supporting optical detection of targets suspended, fixed, or immobilized within the matrix.

[0189] In variations, the collecting container(s) can include any suitable number of containers with desired volumetric capacities. In examples, the collecting container(s) 420 can be configured with one or more of the following formats: 0.2 mL tube format, 0. 2mL PCR tube format, PCR plate format, strip tubes (e.g., 8x strip tube format), microtiter plate format (e.g., 96-well plate format, 48-well plate format, 24-well plate format, 12-well plate format, etc.), 1.5 mL to 5 mL tube format, conical tube format (e.g., 15 mL-50 mL conical tube format, etc.), test tube format, blood collection tube format, cuvette format, cell culture plate format, other laboratory reaction vessel, or another suitable format. The collecting container(s) can be disposable or reusable.

[0190] As such, the system 400 can include elements and support 3D imaging of cross sections of collecting containers having thicknesses (e.g., wall thicknesses, cross section thicknesses) less than 0.1 mm, less than 0.2 mm, less than 0.5 mm, less than 1 mm, less than 2 mm, less than 3 mm, less than 4 mm, less than 5 mm, less than 6 mm, less than 7 mm, less than 8 mm, less than 9 mm, less than 10 mm, less than 15 mm, less than 20 mm, less than 30 mm, less than 40 mm, less than 50 mm, or of other suitable dimensions. The system 400 can additionally or alternatively include elements and support 3D imaging of cross sections of collecting containers having thicknesses (e.g., wall thicknesses, cross section thicknesses) greater than 0. 1 mm, greater than 0.2 mm, greater than 0.5 mm, greater than 1 mm, greater than 2 mm. greater than 3 mm, greater than 4 mm, greater than 5 mm, greater than 6 mm, greater than 7 mm, greater than 8 mm, greater than 9 mm, greater than 10 mm, greater than 15 mm, greater than 20 mm, greater than 30 mm, greater than 40 mm, greater than 50 mm, or of other suitable dimensions.

[0191] The system 400 can, however, include other suitable elements for suspending / immobilizing targets, and detecting targets of a sample in a desired manner.Atty. DocketNo.: 43161-64728 / WO (003WO)

[0192] Aspects of methods performed by embodiments, variations, and examples of the system 400 are described in more detail herein.6.3. Methods

[0193] As shown in FIG. 8A, an embodiment of a method 200 for generation of partitions can include: generating a plurality of partitions within a collecting container at a high rate, each of the plurality of partitions including an aqueous mixture for a digital analysis S210. In embodiments, upon generation, the plurality of partitions is stabilized in position in a close- packed format (e.g., three-dimensional close-packed format, hexagonal close-packed format, rectangular close-packed format, etc.) within a continuous phase, within a region of the collecting container S220, as a partition matrix.

[0194] As shown in FIG. 8B, additional steps of a method 200 for immobilization of partitions of a partition matrix can include: immobilizing positions of a set of partitions of a partition matrix (e.g., within a container, within a collecting container, at a substrate) S270 upon: transmitting a polymer material in a flow state into the collecting container and over the partition matrix S280; and transitioning the polymer matenal to a set state within the collecting container S290, thereby positionally-stabilizing or fixing positions of the partition matrix. In relation to optical clarity characteristics described, the partition matrix can have optical clarity above a threshold level of clarity (and be an emulsion); and transitioning the polymer material to a set state within the collecting container in Step S290 maintains optical clarity of the partition matrix above a threshold level of clarity. In some embodiments, the partition matrix comprises a set of aqueous partitions generated from a sample, wherein the set of aqueous partitions is stabilized in position in a close-packed format within an aqueous continuous phase, and wherein each of the set of aqueous partitions comprises a thin film (or other layer) that is immiscible with the aqueous continuous phase. In some embodiments, each partition of the set of partitions of the partition matrix is surrounded by the polymer material. In some embodiments, the polymer material is provided at a surface of the partition matrix as a cap. In some embodiments, the polymer material comprises a thermoplastic polymer material. In some embodiments, the thermoplastic polymer material comprises agarose. In some embodiments, the polymer material is in a form of a polymer materialcontaining solution. In some embodiments, the polymer material comprises a thermosetting polymer material. In some embodiments, transmitting the polymer material in the flow state comprises heating the polymer material prior to transmission into the collecting container. In some embodiments, the flow state is a continuous flow state. In some embodiments,Atty. DocketNo.: 43161-64728 / WO (003WO) transitioning the polymer material to the set state comprises centrifuging the collecting container in coordination with cooling of the polymer material within the collecting container. In some embodiments, transitioning the polymer material to the set state comprises cross-linking the polymer material. In some embodiments, the set of partitions of the partition matrix contains a set of targets. In some embodiments, each of the set of partitions contains at most one target of the set of targets. In some embodiments, the set of targets comprises a set of nucleic acid targets. In some embodiments, the set of nucleic acid targets comprises a set of nucleic acid targets encoding a set of protein targets. In some embodiments, the set of targets comprises a set of protein targets. In some embodiments, the partition matrix with the polymer material has optical clarity above the threshold level of clarity without use of refractive index matching between the partition matrix and the polymer material. In some embodiments, the threshold level is at least 80% transmissivity of light. In some embodiments, the polymer material-containing solution comprises 0.02 to 3% w / v% of agarose. In some embodiments, the polymer material-containing solution comprises at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0. 1%. 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.1%, at least 1.2%, at least 1.3%, at least 1.4%, at least 1.5%, at least 1.6%, at least 1.7%, at least 1.8%, at least 1.9%, at least 2%, at least 2. 1%, at least 2.2%. at least 2.3%, at least 2.4%, at least 2.5%, at least 2.6%. at least 2.7%, at least 2.8%, at least 2.9%, at least 3%. or greater (in w / v%) of agarose. In some embodiments, the polymer material -containing solution comprises up to 0.02%, up to 0.03%, up to 0.04%, up to 0.05%, up to 0.06%, up to 0.07%, up to 0.08%, up to 0.09%, up to 0.1%, 0.2%, up to 0.3%, up to 0.4%, up to 0.5%, up to 0.6%, up to 0.7%, up to 0.8%, up to 0.9%, up to 1%, up to 1.1%. up to 1.2%, up to 1.3%, up to 1.4%, up to 1.5%, up to 1.6%, up to 1.7%, up to 1.8%, up to 1.9%, up to 2%, up to 2.1%, up to 2.2%, up to 2.3%, up to 2.4%, up to 2.5%, up to 2.6%, up to 2.7%, up to 2.8%, up to 2.9%, up to 3%, or greater (in w / v%) of agarose. In some embodiments, the polymer material-containing solution comprises from about 0.02 to about 0.1% w / v%, from about 0.1 to about 0.25% w / v%. from about 0.25 to about 0.5% w / v%, from about 0.5 to about 0.75% w / v%, from about 0.75 to about 1% w / v%, from about 1 to about 1.25% w / v%, from about 1.25 to about 1.5% w / v%, from about 1.5 to about 1.75% w / v%, from about 1.75 to about 2% w / v%, from about 2 to about 2.25% w / v%, from about 2.25 to about 2.5% w / v%, from about 2.5 to about 2.75% w / v%, from about 2.75 to about 3% w / v%, or any intermediate range (in w / v%) of agarose. In some embodiments, the polymer material-containing solution comprises an agaroseAtty. DocketNo.: 43161-64728 / WO (003WO) solution (e.g., containing an amount of agarose as disclosed herein) in 20 to 100% of a continuous phase described herein. In some embodiments, the polymer material-containing solution comprises 0.02 to 3% w / v% of an agarose solution in 20 to 100% of the continuous phase described herein.

[0195] In some embodiments, the polymer material can be combined with one or more additional components that provide functionality’ to the polymer, where the additional component(s) can diffuse or otherwise be transmitted into the partitions of the partition matrix (e.g., in order to interact with targets captured in the partitions of the partition matrix). Embodiments of the additional component(s) can include one or more of: chemical probes, affinity ligands, enzymatic components, dyes (e.g., SYBR Green, hydrolysis probes, nonhydrolysis probes, cell viability-associated dyes, etc.), sequence-specific capture agents (e.g., antisense oligonucleotides, LNA-modified probes, PNA clamps, morpholino oligomers, etc.), CRISPR guide RNAs, molecular beacons, enzymes (e.g., polymerases, reverse transcriptases, ligases, nickases, recombinases, strand-displacing polymerases, etc ), small molecules (e.g., bis-intercalators (e.g., YOYO-1), minor-groove binders (e.g., netropsin, distamycin). G- quadruplex stabilizers (e.g., TMPyP4, BRACO-19), etc.), cell-penetrant viability dyes (calcein-AM, SYTO dyes), membrane-impermeant exclusion dyes (7-AAD, TO-PRO-3), lectins, aptamers, antibody-conjugated fluorophores, and metabolic reporters (resazurin, tetrazolium salts), metal-chelating probes, click-chemistry tags, barcoding oligos, lysis reagents, permeabilization reagents, fixing reagents, and / or other materials.

[0196] Embodiments of the method 200 can function to generate a plurality of partitions at a high and unprecedented rate in the context of digital analyses and other assays, where the partitions are stabilized in position (e.g., in a close-packed format, in equilibrium stationary' positions) as a partition matrix within a collecting container. Embodiments of the method 200 may further function to reliably generate partitions in a consistent and controlled manner (e.g., as monodisperse and uniform partitions having little-to-no poly dispersity) for various applications, such as digital amplification and analysis and other assays; capture of target material at cellular, subcellular, and molecular scales; sample analysis benefitting from partitions generation; and / or other suitable applications. Embodiments of the method may function to generate partitions using devices that are non-microfluidic, disposable or reusable, in a cost-effective manner.

[0197] Embodiments, variations, and examples of the method 200 can be implemented by or by way of embodiments, variations, and examples of components of the system 100Atty. DocketNo.: 43161-64728 / WO (003WO) described herein. However, the method 200 can additionally or alternatively be configured to perform other suitable methods.

[0198] As shown in FIG. 12A, an embodiment of a method 500 can include: fixing positions of a set of targets of a sample within a matrix in three dimensions S510, wherein the matrix has a level of optical clarity greater than a threshold level (e.g., a threshold level that is at least 80% transmissivity of light), wherein fixing positions comprises: combining the sample with a set of processing reagents for a reaction, and a gelling material at a temperature above a melting temperature of the gelling material, and reducing the temperature of the sample, the set of processing reagents, and the gelling material below the melting temperature, thereby generating the matrix; linking amplified targets of the set of targets with a set of probes of the set of processing reagents upon performing the reaction S520; detecting signals emitted from probes of the set of probes associated with targets of the set of targets upon scanning the matrix w ith an optical detection system S530; and generating a characterization of the set of targets of the sample from the detected signals S540. In some embodiments, the set of targets comprises a set of nucleic acid targets. In some embodiments, the gelling material comprises at least one of agarose, carrageenan, polyethyleneglycol diacrylate (PEGDA), and polyacrylamide. In some embodiments, the set of processing reagents comprises a set of probes configured to associate with targets of the set of targets upon performing the reaction and to emit fluorescent signals upon associating w ith targets of the set of targets. In some embodiments, the method further comprises crosslinking the gelling material prior to performing the reaction. In some embodiments, performing the reaction comprises performing the reaction below the melting temperature of the gelling material. In some embodiments, the reaction comprises an isothermal amplification reaction. In some embodiments, the reaction comprises a rolling circle amplification reaction that physically links amplicons of a target template to the target template. In some embodiments, the method further comprises restricting movement of amplicons of a target template aw ay from the target template. In some embodiments, restricting movement comprises coupling amplicons of the target template to a region of the matrix in proximity to the target template. In some embodiments, restricting movement comprises confining amplicons of the target template within a membrane. In some embodiments, generating the characterization comprises one or more of: generating a count of the set of targets; determining co-occurrence of markers associated with each of a set of different analy te types; characterizing changes in detected signals over a set of time points; and characterizing changes in detected signals in response to a stimulus. In some embodiments, generating the characterization comprises generating aAtty. DocketNo.: 43161-64728 / WO (003WO) count of the set of targets. In some embodiments, generating the characterization comprises determining co-occurrence of markers associated with each of a set of different analyte types. In some embodiments, generating the characterization comprises characterizing changes in detected signals over a set of time points. In some embodiments, generating the characterization comprises characterizing changes in detected signals in response to a stimulus. In some embodiments, the threshold level is at least 80% transmissivity of light.

[0199] As shown in FIG. I2B, a variation of a method 600 can include: generating a count of a set of targets stabilized in position (e.g., random positions, non-random positions, preselected positions, etc.) in three dimensions within a matrix having a level of optical clarity greater than a threshold level S610. The threshold level can be at least 80% transmissivity of light or another suitable level. The set of targets can include greater than 1000 targets. The method 600 can include: linking a set of probes with amplicons of the set of targets upon performing a reaction S620 below the melting temperature within the matrix; and detecting fluorescent signals emitted from the set of probes upon scanning a set of cross sections through the matrix with a light sheet system S630.

[0200] Embodiments, variations, and examples of the methods 500 and 600 can be implemented by or by way of embodiments, variations, and examples of components of the system 400 described herein. However, the methods 500 and 600 can additionally or alternatively be configured to perform other suitable methods.6.3.1. Methods - Matrix Generation

[0201] Step S510 recites: fixing positions of a set of targets of a sample within a matrix in three dimensions S510, wherein the matrix has a level of optical clarity greater than a threshold level. Step S510 functions to spatially suspend and / or immobilize a set and / or distribution of a targets of a sample in a medium that has clarity’ above a threshold level of clarity, for subsequent interrogation (e.g., after a reaction is performed). In embodiments, variations, and examples, the set and / or distribution of targets can include one or more of: nucleic acid targets, protein targets, viral particles, exosomes. cells, cell parts, and / or any combination of various target types, where various target types are described herein. In some embodiments, the set and / or distribution of targets comprises a set and / or distribution of nucleic acid targets. In some embodiments, the set and / or distribution of nucleic acid targets comprises a set and / or distribution of nucleic acid targets encoding a set and / or distribution of protein targets. In some embodiments, the set and / or distribution of targets comprises a setAtty. DocketNo.: 43161-64728 / WO (003WO) and / or distribution of protein targets. In some embodiments, the set and / or distribution of targets comprises a set and / or distribution of single cells.

[0202] In variations, fixing positions can include: combining a sample with a set of processing reagents for a reaction, and a gelling material at a temperature above a melting temperature of the gelling material, and reducing the temperature of the sample, the set of processing reagents, and the gelling material below the melting temperature, thereby generating the matrix. In some embodiments, the melting temperature of the gelling material is at least 40 °C, at least 45 °C, at least 50 °C, at least 55 °C, at least 60 °C, at least 65 °C, at least 70 °C, at least 75 °C, at least 80 °C, at least 85 °C, at least 90 °C, at least 95 °C, at least 100 °C, at least 105 °C, at least 110 °C, at least 115 °C, at least 120 °C. at least 125 °C, at least 130 °C. at least 135 °C. at least 140 °C, at least 145 °C, at least 150 °C, any other intermediate value, or greater. In some embodiments, the melting temperature of the gelling material is 40 °C or lower, 45 °C or lower, 50 °C or lower, 55 °C or lower, 60 °C or lower, 65 °C or lower, 70 °C or lower, 75 °C or lower, 80 °C or lower, 85 °C or lower, 90 °C or lower, 95 °C or lower, 100 °C or lower, 105 °C or lower. 110 °C or lower. 115 °C or lower. 120 °C or lower, 125 °C or lower, 130 °C or lower, 135 °C or lower, 140 °C or lower, 145 °C or lower, 150 °C or lower, any other intermediate value, or lower. In variations, the gelling material can be a UCST material or a LCST material. In variations, generating the matrix can include providing the gelling material at a temperature below a critical temperature of the gelling material, and increasing the temperature of the sample, the set of processing reagents, and the gelling material above the critical temperature.

[0203] In embodiments, variations, and examples, the matrix can be gel-like, where examples of a gel-like matrix include a hydrogel matrix. Material of the matrix can be provided in a liquid state (e.g.. at a temperature above the melting temperature), a solid state (e.g., at a temperature below the melting temperature), or a transition state between a liquid state and a solid state. Hydrogels can be stimulus-responsive (e.g., temperature responsive, pH responsive electric field responsive, light-responsive, etc.). While upper critical solution temperature (UCST) hydrogels are described, the hydrogels can alternatively be lower critical solution temperature (LCST) hydrogels that transition from a liquid to a solid as the temperature increases (e.g., as in a pluronic material). The matrix may comprise a hydrogel material. In some embodiments, the matrix comprises a hydrogel material. In some embodiments, the matrix does not comprise a hydrogel material.

[0204] In embodiments, variations, and examples of Step S510, the gelling material of the matrix 410 can include at least one of agarose, gelatin, carrageenan, polyacrylamide,Atty. DocketNo.: 43161-64728 / WO (003WO) polyethyleneglycol (PEG), polyethyleneglycol diacrylate (PEGDA), gellan gum, agar, or other suitable gelling material. In some embodiments, the gelling material of the matrix includes agarose. In some embodiments, the gelling material of the matrix includes gelatin. In some embodiments, the gelling material of the matrix includes carrageenan. In some embodiments, the gelling material of the matrix includes polyacrylamide. In some embodiments, the gelling material of the matrix includes polyethyleneglycol (PEG). In some embodiments, the gelling material of the matrix includes polyethyleneglycol diacrylate (PEGDA). In some embodiments, the gelling material of the matrix includes gellan gum. In some embodiments, the gelling material of the matrix includes agar. However, other gelling materials that have suitable melting temperature properties, optical clarity characteristics, and characteristics that do not result in inhibiting reaction performance, can be used.

[0205] In variations, step S510 can include mixing a sample comprising targets and processing reagents with material of the matrix in a precursor form (e.g., liquid form at a first temperature) and then transitioning the material of the matrix to a second form (e.g., solid form at a second temperature, upon crosslinking, etc.). After distributing the set of targets of the sample throughout the matrix, the matrix can be crosslinked (e.g., photo-crosslinked, chemically crosslinked, crosslinked with a pH shift, crosslinked with a temperature shift, physically crosslinked, or crosslinked in another suitable manner). The matrix maybe crosslinked. In some embodiments, the matrix is crosslinked. As such, the method can further include crosslinking the gelling material prior to performing the reaction in subsequent steps. In some embodiments, the matrix is not crosslinked.

[0206] In an alternative embodiment, each analyte unit (e.g., molecule, cell, nucleus, other unit) can be isolated in a sub-matrix component (e.g., gel, bead, etc.) and then aggregated together (e.g., container), followed by adding a gelling material or reagent to form a bulk matrix.

[0207] In an alternative embodiment, the set of targets can be stabilized in position within a fluid, such as a density gradient suspending solution that reduces or eliminates mobility' of the set of targets (e.g., during scanning or optical interrogation).

[0208] In variations, the sample fluid can include a sample with a set of targets and materials for an amplification reaction. In a specific example, the sample fluid can be an aqueous mixture including: Nuclease-free water, an amplification master mixture, probes for tagging of targets of the sample, and primers.

[0209] Sample fluids and / or other fluids associated with the matrix can further include materials described in U.S. Pat. No. 11,162,136 granted on 02-NOV-2021, incorporated byAtty. DocketNo.: 43161-64728 / WO (003WO) reference herein. As such, the matrix can have a high degree and greater than a threshold level of clarity, with or without refractive index matching. In variations, the threshold level of clarity of the matrix is associated with a transmissivity greater than 50% transmissivity, greater than 60% transmissivity, greater than 65% transmissivity, greater than 70% transmissivity, greater than 75% transmissivity, greater than 80% transmissivity, greater than 85% transmissivity, greater than 90% transmissivity, greater than 95% transmissivity, greater than 99% transmissivity, etc., upon measuring clarity of the matrix using a transmission detector. In variations, the threshold level of clarity of the matrix is associated with a transmissivity of at least 50% transmissivity, at least 60% transmissivity, at least 65% transmissivity', at least 70% transmissivity, at least 75% transmissivity, at least 80% transmissivity, at least 85% transmissivity, at least 90% transmissivity, at least 95% transmissivity, at least 99% transmissivity, etc., upon measuring clarity of the matrix using a transmission detector.6.3.2. Methods - Target Labeling Reactions

[0210] Step S520 recites: linking amplified targets of the set of targets with a set of probes of the set of processing reagents upon performing the reaction. Relatedly, in relation to method 600, Step S620 recites: linking a set of probes with amplicons of the set of targets upon performing a reaction (e.g., at a temperature appropriate to the critical temperature within the matrix). Steps S520 and S620 function to label (directly or indirectly) the set of targets with markers that can be detected, from within the matrix, due to the optical characteristics of the matrix, as described herein.

[0211] Exemplary reactions can include one or more of: nucleic acid amplification (e g., by polymerase chain reaction (PCR) methods, by isothermal methods (i.e., such that the reaction is an isothermal amplification reaction) such as loop-mediated isothermal amplification (LAMP), by recombinase polymerase amplification (RPA), by multiple displacement amplification (MDA), by helicase dependent amplification (HDA), by strand displacement amplification (SDA), by nicking enzyme amplification (NEAR), by transcription mediated amplification (TMA), by digital helicase dependent amplification, by RNaseH mediated amplification, by whole genome amplification (WGA), by rolling circle amplification, etc.) on purified DNA, cDNA, RNA, or directly from lysate (e.g., blood lysate); fluorescent in situ hybridization (FISH) with fluorescently tagged nucleic acids (e.g., PNA, LNA, DNA, RNA, etc.) or an indirect in situ hybridization approach using DIG or biotin; by an in vitro transcription or translation assay (e.g., whereby a colorimetric or fluorescent reporter is usedAtty. DocketNo.: 43161-64728 / WO (003WO) for detection); partition PCR applied to samples derived from single cells (e.g., prokary otes, eukaryotes), organelles, viral particles, and exosomes; droplet analysis of proteins (e.g., by proximity ligation assays, etc.): sequencing applications (e.g., single molecule sequencing applications); monitoring or detection of products (e.g., proteins, chemicals) released from single cells (e.g., interleukin released from immune cells); monitoring cell survival and / or division for single cells; monitoring or detection of enzymatic reactions involving single cells; target material capture at cellular (e.g., mammalian cell, bacterial cell, pathogen, viral, etc.) and sub-cellular (e.g., organelle, molecular, etc.) scales; enumeration of heterogeneous cell populations in a sample; enumeration of individual cells or viral particles (e.g., by encapsulating cells in droplets with species-specific antibodies coupled with enzy mes that react with substrate components in the droplet to produce signals, etc.); monitoring of viral infections of a single host cell; and other suitable applications.

[0212] In embodiments, variations, and examples, the set of processing reagents can include a set of probes configured to associate with targets and / or amplicons of the set of targets upon performing the reaction and to emit fluorescent signals upon associating with targets and / or amplicons of the set of targets. As such, the targets can be directly or indirectly fluorescently- labeled by the set of probes. The set of probes can include sequences configured to interact with target sequences, sequences complementary7to target sequences, and / or adapter sequences associated with target sequences (e g., where adapters can be added during amplification reactions). The set of probes can include dyes that emit a signal when associated with target sequences, and the signals can be detected with amplification of target sequences. In some embodiments, association and / or interaction of the set of probes of the set of processing reagents with targets and / or amplicons of the set of targets comprises hybridization (e.g.. based on complementary7sequences). In some embodiments, each probe of the set of probes is configured to emit a fluorescent signal.

[0213] In examples, dyes (e.g., for tagging of RNAs, DNAs, oligonucleotides, etc.) can include one or more of: FAM, (e.g., 6-FAM), Cy3TM, Cy 5TM, Cy5.5TM, TAMRATM (e.g., 5-TAMRA, 6-TAMRA, etc ), MAX, JOE. TETTM. ROX, TYETM (e.g., TYE 563, TYE 665, TYE 705. etc.). Yakima Yellow ®. HEX, TEX (e.g.. TEX 615). SUN. ATTOTM (e.g.. ATTO 488, ATTO 490LS, ATTO 532, ATTO 550, ATTO 565, ATTO RholOl, ATTO 590, ATTO 633, ATTO 647, ATTO 647N, etc.), Alexa Fluor ® (e.g., Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 750, etc.), IRDyes® (e.g., 5’IRDye 700. 5'IRDye 800, 5’IRDye 800CW. etc.), Rhodamine (e.g., Rhodamine Green, Rhodamine Red, Texas Red ®, Lightcycler ®, Dy 482XL, DyAtty. DocketNo.: 43161-64728 / WO (003WO)508XL, Dy 526XL, Dy 750, Hoechst dyes, DAPI dyes, SYTOX dyes, chromomycin dyes, mithramycin dyes, YOYO dyes, ethidium bromide dyes, acridine orange dyes, TOTO dytes, thiazole dyzes, CyTRAK dyes, propidium iodide dyes, LDS dyes, BODIPY dyes, and / or other dyes. In some embodiments of the systems, methods, devices, and compositions disclosed herein, the dyes used are selected from: Alexa Fluor 488, Alexa Fluor 594, ATTO 490LS. ATTO 532, ATTO 647N, Cy5TM, FAM, Dy 482XL, Dy 508XL, Dy 526XL, and combinations thereof. In examples, cell function dyes for tagging of target material and detection can include one or more of: DCFH, DHR, SNARF, indo-1, Fluo-3, Fluo-4, and / or other dyes. In examples, fluorescent proteins for tagging of target material and detection can include one or more of: cerulean, mCFP, mTurquoise, T-Sapphire, CyPet, ECFP, CFP. EBFP, Azurite, and / or other fluorescent proteins.

[0214] In an embodiment, dyes used for 10-plex or greater multiplexing of targets can include: Alexa Fluor™ 488, Atto™ 532, Alexa Fluor™ 594, Atto™ 647N, Cy5, FAM, Dy- 526XL, Dy-508XL, Dy-482XL, and Atto™ 490LS.

[0215] As such, detection of labeled targets can involve fluorescent nucleic acid probes, protein markers, lipid markers, carbohydrates markers, or other optically-detectable markers.

[0216] Other aspects of the set of processing reagents include embodiments, variations, and examples of processing reagents described in U.S. Application No. 18 / 085,217 filed on 20- DEC-2022, which is herein incorporated in its entirety by this reference.

[0217] In embodiments, steps S510, S520. and S610 can include optically clarifying the matrix upon generation (e.g., during combination with the set of targets and / or the set of processing reagents). Alternatively, steps S510, S520, and S610 can include optically clarifying the matrix at some point after generation, such as after the set of targets and / or the set of processing reagents are dispersed throughout the matrix in solid form, after target amplification, etc. Clarification can involve the use of a refractive index-matching fluid, a clarifying agent that can be applied to the matrix or used to surround the collecting container 420, or another suitable agent.

[0218] In relation to steps S510, S520, and S610, the set of targets can be freely suspended in the matrix or fluid (e.g., fluid with a densify gradient, as described herein). In relation to set and / or distribution of the set of targets in a matrix, the set of targets can be anchored in the matrix by way of crosslinking (e.g. for nucleic acids or other molecules, template molecules can be modified to have a functional group, such as an acrydite group, that crosslinks into hydrogels during polymerization). In a specific example, target nucleic acid molecules can be modified to have an acrydite group that crosslinks with PEGDA during polymerization.Atty. DocketNo.: 43161-64728 / WO (003WO)

[0219] In relation to steps S510, S520, and S610, method steps can further include restricting movement of amplicons of a target template (e.g., template target nucleic acid) away from the target template. Amplicons can thus be physically linked to the target template (e.g. using rolling circle amplification of a circular DNA template, etc.). As such, a reaction within the matrix can include a rolling circle amplification reaction that physically links amplicons of a target template to the target template. Restricting movement can additionally or alternatively include coupling amplicons of the target template to a region of the matrix in proximity to the target template. Restricting movement can additionally or alternatively include physically linking amplicons to the matrix as they are formed (e.g. using primers of the set of processing reagents that have anchors that crosslink with matrix). Restricting movement can additionally or alternatively include confining amplicons of the target template within a membrane. For instance, amplicons of a target template can be confined to within a cell or viral particle due to their biological membranes or cellular structures. In a specific example, gene targets of a suspension of fixed cells be amplified and retained in position within fixed cell membranes (e.g. with performance of a FISH-like reaction in fixed cells to detect if cells carry a mutation or gene target). The amplified targets within membranes can be suspended or immobilized, as described herein, and then optically interrogated. Target amplicons can additionally or alternatively have restricted diffusion away from an originating target template due to the network structure of the matrix 410.

[0220] In relation to steps S520 and S620, performing the reaction can include transmitting heat to and from the matrix (e.g., within the collecting container) during a heat transmission operation. Heat transmission can be associated with cold storage (e.g., refrigeration, freezing, etc.), thermocycling (e.g., during an amplification process), incubation, lysis, enzyme activation, or another heat transmission operation. In variations, the temperature may vary between 0 °C to 95 °C during the heat transmission operation, or another temperature range suited to critical temperatures of the matrix.6.3.3. Methods - Signal Detection

[0221] Step S530 recites: detecting signals emitted from probes of the set of probes associated with targets of the set of targets upon scanning the matrix with an optical detection system. Relatedly, in relation to method 600, Step S630 recites: detecting fluorescent signals emitted from the set of probes upon scanning a set of cross sections through the matrix with a light sheet system.Atty. DocketNo.: 43161-64728 / WO (003WO)

[0222] Steps S530 and S630 function to perform an optical interrogation operation for detection of signals associated with the set and / or distribution of targets from within the matrix (e.g.. within the collecting container), where the optical interrogation operation can include readout of signals (e.g., light signals, fluorescent signals, colorimetric signals, etc.) from the matrix. In particular, readout can be performed for cross sections of the matrix within the collecting container, using techniques described in applications incorporated by reference.

[0223] In variations of steps S530 and S630, readout of fluorescent signals (e g., from labeled targets and / or analytes within the matrix, from products of targets and / or analytes within the matrix, etc.) can be performed by one or more of a 3D scanning technique (e.g., light sheet imaging, confocal microscopy, etc.) and a planar imaging technique (e.g.. to take images of a cross-section of the closed container). Additionally or alternatively, in some applications, readout of colorimetric changes associated with targets and / or analytes within the matrix can be performed by 3D imaging techniques (e.g., 3D brightfield construction using light field imaging, etc.). Readout can be performed for each of a set of cross sections of the matrix, across multiple color channels (e.g., two color channels, three color channels, four color channels, five color channels, six color channels, seven color channels, etc.).

[0224] Readout can be performed for 10 cross-sections of the matrix, 20 cross-sections of the matrix, 30 cross-sections of the matrix, 40 cross-sections of the matrix, 50 cross-sections of the matrix, 60 cross-sections of the matrix, 70 cross-sections of the matrix, 80 crosssections of the matrix, 90 cross-sections of the matrix, 100 cross-sections of the matrix, 200 cross-sections of the matrix, 300 cross-sections of the matrix, 400 cross-sections of the matrix, 500 cross-sections of the matrix, 600 cross-sections of the matrix, any intermediate value, or greater, for each of the set of color channels. Additional aspects of scanning and interrogation of a matrix are described in U.S. Application No. 18 / 583,701 filed on 21-FEB- 2024, which is herein incorporated in its entirety by this reference. Additional aspects of multiplexed assays involving a matrix are described in U.S. Application No. 18 / 085,217 filed on 20-DEC-2022, which is herein incorporated in its entirety by this reference.

[0225] In specific examples, readout associated with digital analyses (e.g., counting, quantification, etc.) for each channel can be performed within a duration of 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, 30 seconds, 20 seconds, 10 seconds, or less, depending upon one or more of signal-to-noise ratio, optical sensor sensitivity, excitation power (e.g., of a light source used to illuminate and induce fluorescence), or other characteristics.Atty. DocketNo.: 43161-64728 / WO (003WO)

[0226] In other variations, readout of non-fluorescent signals from the matrix can be performed. For instance, products resulting from reactions within the partition matrix can produce changes in one or more of refractive indices, light absorption, light scattering, light reflection, light transmission, or other light interaction characteristics that are different from empty or unreacted partitions, for detection by various techniques (e.g., spectrophotometric techniques, turbidimetric techniques, etc.).

[0227] As such, methods described herein enable digital analyses across a wide dynamic range that is 10-100 times greater than that of existing technologies, depending upon application of use. In variations, the methods 500, 600 enable characterization of targets of interest without requiring Poisson statistics-associated correction factors for error.6.3.4. Methods - Target Characterization

[0228] Step S540 recites: generating a characterization of the set of targets of the sample from the detected signals. Relatedly, in relation to method 600, Step S610 recites: generating a count of a set of targets stabilized in position in three dimensions within a matrix.

[0229] As such, upon detection of signals from the set and / or distribution of targets suspended or immobilized in a matrix in relation to steps S530, various analyses characterizing the set of targets can be performed. Generating a characterization of the set of targets can include one or more of: generating a count of the set of targets (e.g., targets labeled by fluorescent markers); determining co-occurrence of markers associated with each of a set of different analyte types (e.g., molecular types, cellular types, etc ); characterizing changes in detected signals over a set of time points (e.g., in relation to monitoring growth of a culture, in relation to monitoring amplification of signals over the span of a reaction, etc.); and characterizing changes in detected signals in response to a stimulus (e.g.. environmental factor, heat stimulus, for melt curve analysis, an electromagnetic stimulus [e.g., gene expression resulting from exposure to an electromagnetic stimulus, uncaging of reaction components by UV light, such as with caged polymerase or dNTPs], a chemical stimulus [e.g., with infusion of chemicals into the matrix that affects signal intensities, with use of growth factors for cells or tissues, with use of a denaturant for DNA or proteins], etc.).

[0230] The methods 500, 600 can be implemented by an embodiment, variation, or example of the system 400 described herein, or can alternatively be implemented by another suitable system.Atty. DocketNo.: 43161-64728 / WO (003WO)6.3.5. Methods - Partition and Partition Matrix Generation

[0231] In relation to generation of partitions at a high rate in Step S210. variations of the method 200 can produce partitions at a rate of at least 50,000 partitions / minute, of at least 100,000 partitions / minute, of at least 200,000 partitions / minute, of at least 300,000 partitions / minute, of at least 400,000 partitions / minute, of at least 500,000 partitions / minute, of at least 600,000 partitions / minute, of at least 700,000 partitions / minute, of at least 800,000 partitions / minute, of at least 900,000 partitions / minute, of at least 1 million partitions / minute, of at least 2 million partitions / minute, of at least 3 million partitions / minute, or greater, using embodiments, variations, and examples of system elements described herein. Partitions can be generated at the high rate, using embodiments, variations, and examples of the membrane(s) 120 described herein, in relation to hole density, hole-to-hole spacing, hole diameter, membrane thickness, hole aspect ratio, membrane material, and / or other characteristics.

[0232] In relation to partition generation in Step S210, a high number of partitions can be generated within a container (e.g., collecting container interfaced with membrane supporting elements described herein), wherein, in variations, greater than 100,000 partitions, greater than 500,000 partitions, greater than 1 million partitions, greater than 2 million partitions, greater than 3 million partitions, greater than 4 million partitions, greater than 5 million partitions, greater than 6 million partitions, greater than 7 million partitions, greater than 8 million partitions, greater than 9 million partitions, greater than 10 million partitions, greater than greater than 15 million partitions, greater than 20 million partitions, greater than 25 million partitions, greater than 30 million partitions, greater than 40 million partitions, greater than 50 million partitions, greater than 100 million partitions, greater than 200 million partitions, greater than 300 million partitions, or greater can be generated within the collecting container.

[0233] In variations, the collecting container can have a volumetric capacity less than 50 microliters or from 50 through 300 microliters and greater. An example of a collecting container can include a PCR strip tube having a volumetric capacity from 20 microliters to 50 microliters; however, other variations and examples of collecting containers are described in more detail herein. Partitions generated in Step S210 may have a characteristic dimension (e.g., from 1-50 micrometers, from 10-30 micrometers, intermediate values within ranges described, greater than 50 micrometers, greater than 30 micrometers, etc.) that is relevant for digital analyses, single cell capture, target detection, individual molecule partitioning, target partitioning or other applications.Atty. DocketNo.: 43161-64728 / WO (003WO)

[0234] Generating the plurality of partitions in Step S210 can include driving a sample fluid through a membrane comprising a distribution of holes, the membrane aligned with or coupled to a reservoir outlet of a reservoir for the sample fluid. Driving the sample fluid can include applying a centrifugal force (e.g., by centrifugation) to drive the sample fluid through the holes of the membrane. In variations, the centrifugal force can be applied at 1,000g, 2,000g. 3,000g, 4,000g, 5,000g, 6,000g, 7,000g. 8,000g, 9,000g, 10,000g, 11,000g, 12.0000g, 13.000g, 14,000g, 15,000g, 16,000g, 17,000g. 18.000g. 19.000g. 20.000g, 30,000g, any intermediate value, or greater than 30,000g. Duration of spinning can be 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes. 20 minutes, 30 minutes, 40 minutes. 50 minutes, any intermediate value, or greater than 50 minutes, where spin duration is a function of the amount of sample fluid being partitioned and used to form partition matrices according to methods described herein.

[0235] In relation to generation of the partition matrix (e.g., by driving sample fluids through one or more immiscible layers of fluid to form a multi-phase emulsion), where resultant emulsions generated have viscous properties, shear-thickening properties, and / or gel-like properties (e.g., as is present when generating partition matrices where partitions of a sample are a first phase surrounded by films (or other layers) of a second phase that is immiscible with the first phase, and the immiscible phase films (or layers) are surrounded by a continuous third phase that is immiscible with the second phase), centrifugation can produce partition matrices having uneven top / superior surface (e.g., surfaces furthest away from a base of the collecting container, along a force axis in a radial direction attributed to centrifugation), an example of which is shown in FIG. 9A. Such uneven surfaces can increase difficulty of readout of signals from partitions near the top / superior surface (e.g.. emulsion surface furthest from the base of the collecting container), due to higher background, reduced clarity, and / or other factors.

[0236] As such, the method 200 can further include S214, which, as shown in FIG. 9B involves spinning the sample fluid, the membrane, and the collecting container within a centrifuge in a first direction of rotation, and reversing the direction of rotation, thereby adjusting a surface profile of an emulsion comprising the plurality of partitions within the collecting container. Adjusting the surface profile can improve one or more of levelness, planarity, or other characteristics of the surface profile to improve readout ability. In relation to S214, spinning in the first direction and the second direction can be performed at centrifugal forces in the ranges provided herein or outside described ranges. Furthermore,Atty. DocketNo.: 43161-64728 / WO (003WO) spinning in the first direction can be performed at a first rotational velocity, and spinning in the second direction can be performed at a second rotational velocity different than the first rotational velocity.

[0237] Additionally or alternatively, the method 200 can further include S216, which as shown in FIG. 9C, involves spinning the sample fluid, the membrane, and the collecting container within a centrifuge at a first rotational velocity and at a second rotational velocity’ less than the first rotational velocity, thereby adjusting a surface profile of an emulsion comprising the plurality of partitions within the collecting container. Adjusting the surface profile can improve one or more of levelness, planarity, or other characteristics of the surface profile to improve readout ability. In relation to S216, spinning at the first rotational velocity and the second rotational velocity can be performed at centrifugal forces in the ranges provided herein or outside described ranges. Furthermore, in relation to Steps S214 and S216, achieving the first rotational velocity and / or the second rotational velocity can be performed with a ramp-up or acceleration rate, in order to improve surface features of the emulsion.

[0238] In alternative variations, the applied force can be associated with an applied pressure, magnetically applied, or otherwise physically applied to drive sample fluid(s) through the membrane(s).

[0239] In relation to components of the sample fluid and / or fluid layers within the collecting container(s) for generation of the partition matrix, the sample fluid and fluid layers within the collecting container can have one or more of a certain density, viscosity, surface tension, aqueous nature, hydrophobicity, immiscibility characteristics, or other characteristics. Fluids implemented can have densities from 1 through 3000 kg / m3and intermediate values, viscosities from 0.001 through 0.1 Ns / m2, and surface tensions of 0.01 through 1 N / m, depending upon application.

[0240] In specific examples, the sample fluid can include a sample and materials for an amplification reaction. In a specific example, the sample fluid can be an aqueous mixture including: Nuclease-free w ater, an amplification master mixture, probes for tagging of targets of the sample, and primers.

[0241] In specific examples, a continuous aqueous phase provided within the collecting container (e.g., before the sample fluid passes through openings of the membrane and into the collecting container) can include one or more of: a surfactant, such as a non-ionic, nondenaturing detergent that is miscible with water (e.g., octylphenoxypolyethoxyethanol, another octylphenol surfactant, another phenol surfactant, etc.); water (e.g.. PCR-certified water), a sugar solution (e.g., D-(+)-Glucose solution); potassium chloride; magnesiumAtty. DocketNo.: 43161-64728 / WO (003WO) chloride; tris-HCl; sodium azide; and other components. In specific examples, the surfactant can have a raw material unit percentage of 0.01% to 0.2%; water can have a raw material unit percentage of 0.5% to 5%; the sugar solution can have a raw material unit percentage of 2% to 10%; potassium chloride can have a raw material unit percentage of 0.05% to 0. 15%; magnesium chloride can have a raw material unit percentage of 0.02% to 0. 15%; tris-HCl can have a raw material unit percentage of 0.08% to 0.25%; sodium azide can have a raw material unit percentage of 0.08% to 0. 18%.

[0242] In specific examples, an immiscible layer provided over the continuous aqueous phase within the collecting container can include one or more of: an oil-miscible surfactant (e.g., a silicone surfactant); and a bubble suppressing surfactant (e.g., Poly dimethylsiloxane, Trimethylsiloxy). In a specific example, the oil-miscible surfactant can have a raw material unit percentage of 0.005% to 0.05%, and the bubble suppressing surfactant can have a raw material unit percentage of 0.5%-2%.

[0243] In specific examples, the sample fluid is provided within a reservoir, where an embodiment, variation, or example of the membrane is provided as an outlet of the reservoir, and a driving solution is provided over the sample fluid within the reservoir. The dnving solution functions to facilitate application of force to the sample fluid to completely drive the sample fluid through the openings in the membrane. As such, the driving solution can contribute to zero sample waste performance of the sy stems and methods described herein (e.g.. zero dead volume, less than 1% dead volume associated with sample waste). The driving solution is preferably immiscible with the sample fluid, but can be otherwise configured. In a specific example, the driving solution can include a bubble suppressing surfactant (e.g., Poly dimethylsiloxane, Trimethylsiloxy). The driving solution can optionally include a dye or other indication component, where the dye or other indication component can be used to provide an indication that the driving solution has been properly delivered to the reservoir over the sample fluid, and, after driving of the sample fluid through the membrane, to indicate that all of the sample has been driven through the membrane appropriately (and none of the driving solution has passed into the collecting container).

[0244] FIG. 9D depicts an example of the driving solution, the sample fluid, the continuous aqueous phase, and the immiscible layer, within a units of an example of the system described herein.

[0245] Sample fluids and / or fluid layers can further include materials described in U.S. Pat. No. 11.162,136 granted on 02 -NOV -2021, incorporated by reference herein. As such, partitions and / or resulting emulsions generated with said partitions can have a high degreeAtty. DocketNo.: 43161-64728 / WO (003WO) and greater than a threshold level of clarity, with or without refractive index matching. In variations, the threshold level of clarity of the emulsion is associated with a transmissivity greater than 50% transmissivity, greater than 60% transmissivity, greater than 65% transmissivity, greater than 70% transmissivity, greater than 75% transmissivity, greater than 80% transmissivity, greater than 85% transmissivity, greater than 90% transmissivity, greater than 95% transmissivity, greater than 99% transmissivity, etc., (e.g., upon measuring clarity of the emulsion using a transmission detectorf In variations, the threshold level of clarity of the emulsion is associated with a transmissivity of at least 50% transmissivity, at least 60% transmissivity, greater than 65% transmissivity, at least 70% transmissivity, greater than 75% transmissivity, at least 80% transmissivity, greater than 85% transmissivity, at least 90% transmissivity, at least 95% transmissivity, at least 99% transmissivity, etc., (e.g., upon measuring clarity of the emulsion using a transmission detector).

[0246] In embodiments, upon generation, the plurality of partitions may be stabilized in position in a close-packed format (e.g., three-dimensional close-packed format, hexagonal close-packed format, rectangular close-packed format, etc.) within a continuous phase, within a region of the collecting container S220. In relation to the membranes described herein, generating the plurality of partitions can include transmitting partitions (e.g., partitions of the sample fluid from the holes of the membrane(s)) toward a closed end of the collecting container, thereby stabilizing the plurality of partitions, once they pass into fluid layers in the collecting container, in a three dimensional close-packed format toward the closed end of the collecting container. Alternatively, the plurality of partitions of the partition matrix can be stabilized (e.g., within a continuous phase, within a matrix positioned within the collecting container, within a mesh within the collecting container, etc.) toward the closed end or a different region of the collecting container, in a non-close-packed format. For instance, nonclose packed partitions or partitions that can move relative to each other within the closed collecting container can still be processed by optical interrogation as described herein (e.g., by fixing a position of the closed collecting container relative to a scanning path of an optical interrogation instrument). Additionally or alternatively, in relation to close-packed or non- close-packed formats, partitions of a partition matrix can be stabilized in position by curing (e.g., with light, with heat, with a pH shift, with other cross-linking, by way of an electric field, by way of a magnetic field, etc.) the dispersed phase, continuous phase, or both of the emulsion. Still alternatively, partitions may not be stabilized in position or in a close-packed format (e.g., partitions can move relative to each other within a container, such as for water- in-oil or oil-in-water emulsions, etc.).Atty. DocketNo.: 43161-64728 / WO (003WO)

[0247] In some variations, as shown in FIG. 8A, the method 200 can further include: transmitting heat to and from the partitions of the partition matrix, within the collecting container, during a heat transmission operation S230. Heat transmission can be associated with cold storage (e.g., refrigeration, freezing, etc.), thermocy cling (e.g., during an amplification process), incubation, lysis, enzyme activation, or another heat transmission operation. In variations, the temperature may vary between 0 °C to 95 °C during the heat transmission operation, and in specific examples, the temperature can transition between temperatures within the ranges described, with stability of partitions maintained. In particular, given the partition generation techniques and materials described herein, individual partitions of the plurality of partitions remain unmerged with adjacent partitions in the close-packed format during the heat transmission operation.

[0248] In some variations, as shown in FIG. 8A, the method 200 can further include: performing an optical interrogation operation w ith the plurality of partitions within the collecting container S240, where the optical interrogation operation can include readout of signals (e.g., light signals, fluorescent signals, colorimetric signals, etc.) from partitions of the plurality of partitions. In particular, readout can be performed for cross sections of the plurality of partitions within the collecting container, using techniques described in applications incorporated by reference.

[0249] In variations, readout of fluorescent signals (e.g., from labeled analytes within partitions of the partition matrix, from products of analytes within partitions of the partition matrix, etc.) can be performed by one or more of a 3D scanning technique (e.g., light sheet imaging, confocal microscopy, etc.) and a planar imaging technique (e.g., to take images of a cross-section of the closed container). Additionally or alternatively, in some applications, readout of colorimetric changes associated with partitions of the partition matrix can be performed by 3D imaging techniques (e.g., 3D brightfield construction using light field imaging, etc.). Readout can be performed for each of a set of cross sections of the plurality' of partitions / collecting container, across multiple color channels (e.g., two color channels, three color channels, four color channels, five color channels, six color channels, seven color channels, etc.).

[0250] Readout can be performed for 10 cross-sections of the partition matrix, 20 crosssections of the partition matrix, 30 cross-sections of the partition matrix, 40 cross-sections of the partition matrix, 50 cross-sections of the partition matrix, 60 cross-sections of the partition matrix, 70 cross-sections of the partition matrix, 80 cross-sections of the partition matrix, 90 cross-sections of the partition matrix, 100 cross-sections of the partition matrix,Atty. DocketNo.: 43161-64728 / WO (003WO)200 cross-sections of the partition matrix, 300 cross-sections of the partition matrix, 400 cross-sections of the partition matrix, 500 cross-sections of the partition matrix, 600 crosssections of the partition matrix, any intermediate value, or greater, within the closed collecting container, for each of the set of color channels. Additional aspects of scanning and interrogation of partitions of a partition matrix are described in U.S. Application No. 18 / 583,701 filed on 21-FEB-2024, which is herein incorporated in its entirely by this reference. Additional aspects of multiplexed assays involving a partition matrix are described in U.S. Application No. 18 / 085,217 filed on 20-DEC-2022, which is herein incorporated in its entirety by this reference.

[0251] In specific examples, readout associated with digital analyses (e.g., counting, quantification, etc.) for each channel can be performed within a duration of 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, 30 seconds, 20 seconds, 10 seconds, or less, depending upon one or more of signal-to-noise ratio, optical sensor sensitivity, excitation power (e.g., of a light source used to illuminate partitions and induce fluorescence), or other characteristics.

[0252] In other variations, readout of non-fluorescent signals from partitions of the partition matrix can be performed. For instance, products resulting from reactions within individual partitions of the partition matrix can produce changes in one or more of refractive indices, light absorption, light scattering, light reflection, light transmission, or other light interaction characteristics that are different from empty or unreacted partitions, for detection by various techniques (e.g., spectrophotometric techniques, turbidimetric techniques, etc.).

[0253] As such, methods described herein enable digital analyses across a wide dynamic range that is 10-100 times greater than that of existing technologies, depending upon application of use. In examples related to nucleic acid counting, the methods disclosed herein can have a dynamic range from 1 through 100 million, due to the extremely high number of uniform partitions generated from which signals can be read, and due to the ability to partition with low occupancy (e.g., less than 20% occupancy, less than 15% occupancy, less than 10% occupancy, less than 9% occupancy, less than 8% occupancy, less than 7% occupancy, less than 6% occupancy, less than 5% occupancy, etc.) of partitions by targets. In variations, such low occupancy can enable characterization of targets of interest without requiring Poisson statistics-associated correction factors for partitioning error or other error.

[0254] In examples, generation of large numbers of partitions (as described herein) within a closed container can be performed within durations and at rates described herein (e.g., on the order of 1 million partitions / minute), and readout of each channel for a digital analysis can beAtty. DocketNo.: 43161-64728 / WO (003WO) performed at a high rate (e.g., less than 1 minute per channel, across multiple color channels), thereby enabling readout for a digital analysis of millions of partitions on the order of minutes or hours (with time durations as described herein).

[0255] As such, methods for partition generation through readout of numbers of partitions described, for each of a set of channels for a digital analysis, can be performed within a duration of 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, or any intermediate value. In examples, once partition matrix generation and amplification / tagging have been performed, readout of signals from each channel can be performed within 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, 30 seconds, 20 seconds, 10 seconds, or less, depending upon one or more of signal- to-noise ratio, optical sensor sensitivity, excitation power (e.g., of a light source used to illuminate partitions and induce fluorescence), or other characteristics.

[0256] As such, methods for partition generation through readout of numbers of partitions can include: performing a digital analysis of target nucleic acid material from a sample within a duration (e.g., a duration described herein), wherein performing the digital analysis includes: generating a plurality of partitions of a partition matrix (e.g., within a closed collecting container), the plurality of partitions comprising a number of partitions described generated from a combination of the sample and materials for an amplification reaction, individually isolating the plurality of partitions , receiving heat (e.g., through the closed collecting container), thereby amplifying said target nucleic acid material, and transmitting signals (e.g., from a set of cross-sections of the partition matrix comprising the plurality of partitions within the closed collecting container), for readout using a set of channels of a detection system (e.g., a detection system interacting with the closed collecting container).6.3.6. Methods - Partition Matrix Immobilization

[0257] As shown in FIG. 8B. additional steps of a method 200 for fixation / immobilization of positions of a set of partitions of a partition matrix can include; immobilizing positions of a partition matrix (e.g., within a container, within a collecting container, at a substrate) S270 upon: transmitting a polymer material in a flow state into the collecting container and over the partition matrix S280; and transitioning the polymer material to a set state within the collecting container S290, thereby positionally-stabilizing or fixing positions of the partition matrix. In relation to optical clarity characteristics described, the partition matrix can haveAtty. DocketNo.: 43161-64728 / WO (003WO) optical clarity above a threshold level of clarity (and be an emulsion); and transitioning the polymer material to a set state within the collecting container in Step S290 maintains optical clarity of the partition matrix above a threshold level of clarity. In some embodiments, the partition matrix comprises a set of aqueous partitions generated from a sample, wherein the set of aqueous partitions is stabilized in position in a close-packed format within an aqueous continuous phase, and wherein each of the set of aqueous partitions comprises a thin film (or other layer) that is immiscible with the aqueous continuous phase. In some embodiments, each partition of the set of partitions of the partition matrix is surrounded by the polymer material. In some embodiments, the polymer material is provided at a surface of the partition matrix as a cap. In some embodiments, the polymer material comprises a thermoplastic polymer material. In some embodiments, the thermoplastic polymer material comprises agarose. In some embodiments, the polymer material is in a form of a polymer materialcontaining solution. In some embodiments, the polymer material -containing solution comprises 0.02 to 3% w / v% of agarose. In some embodiments, the polymer material comprises a thermosetting polymer material. In some embodiments, transmitting the polymer material in the flow state comprises heating the polymer material prior to transmission into the collecting container. In some embodiments, the flow state is a continuous flow state. In some embodiments, transitioning the polymer material to the set state comprises centrifuging the collecting container in coordination with cooling of the polymer material within the collecting container. In some embodiments, transitioning the polymer material to the set state comprises cross-linking the polymer material. In some embodiments, the set of partitions of the partition matrix contains a set of targets. In some embodiments, each of the set of partitions contains at most one target of the set of targets. In some embodiments, the set of targets comprises a set of nucleic acid targets. In some embodiments, the set of nucleic acid targets comprises a set of nucleic acid targets encoding a set of protein targets. In some embodiments, the set of targets comprises a set of protein targets. In some embodiments, the partition matrix with the polymer material has optical clarity above the threshold level of clarity without use of refractive index matching between the partition matrix and the polymer material. In some embodiments, the threshold level is at least 80% transmissivity of light.

[0258] Applications of fixing and / or otherwise immobilizing generated partitions can function to provide calibration samples (e.g., samples where partitions for calibrating interrogation instruments are fixed). Such calibration samples can reduce computational burden associated with calibration of instruments used to interrogate / scan processed samples while running assays, given that no additional algorithmic adjustments would be necessary' toAtty. DocketNo.: 43161-64728 / WO (003WO) run these calibration samples, given that the calibration samples would be more representative of test samples being processed. Calibration of instruments using such calibration samples can include calibration for one or more of magnification operations, focus operations, thickness characterization (e.g., of scanning slices), uniformity characterization (e.g., of partitions), and power calibration (e.g., of light sheet scanning light emission elements). Applications of fixing and / or otherwise immobilizing generated partitions can function to provide a mechanism by which samples processed remotely can be transported (e.g., shipped) to a test site (e.g., central test site) for sample processing or troubleshooting. Applications of fixing and / or otherwise immobilizing generated partitions can thus also function to increase user accessibility to high performance testing of samples, without requiring users to have an instrument for interrogating / scanning processed samples.

[0259] Step S280 recites: transmitting a polymer material in a flow state into the collecting container and over the partition matrix, which functions to process the partition matrix w ith a material that can be used to immobilize the partition matrix. The polymer material can provide a cap (e.g., a plug) over the partition matrix to immobilize the partition matrix after Step S290 is performed. Additionally or alternatively, the polymer material can permeate the partition matrix and immobilize the partition matrix after Step S290 is performed, such that each partition of the partition matrix is surrounded by the polymer material.

[0260] In variations, the polymer material can include linear polymers and / or branched polymers. In variations, the polymer material can be thermoplastic or thermosetting. In variations, the polymer material can be crosslinkable and / or non-crosslinkable. In variations, the polymer material can be a hydrogel. In variations, the polymer material can be a natural polymer material or a synthetic polymer material. Variations of Step S280 can involve polymer materials that can be transitioned between flow states and set states, according to Step S290, including one or more of: agarose, polystyrene, polystyrene-divinylbenzene, polymethylmethacrylate (PMMA), acrylamide, polyacrylamide, carrageenan, gelatin, polyethylene glycol (PEG), polyethyleneglycol diacrylate (PEGDA), alginate, polyethylene terephthalate, cyclic olefin polymer, cyclic olefin copolymer, styrene- acrylonitrile, polyvinyl butyral, polyethylene naphthalate, polyvinyl alcohol, polylactic acid, epoxy, poly(allyl diglycol carbonate), and other suitable polymer materials. In a specific example, the polymer material can include agarose (e.g., an agarose material). In some embodiments, the polymer material comprises polystyrene. In some embodiments, the polymer material comprises polystyrene-divinylbenzene. In some embodiments, the polymer material comprises polymethylmethacrylate (PMMA). In some embodiments, the polymer material comprisesAtty. DocketNo.: 43161-64728 / WO (003WO) acrylamide. In some embodiments, the polymer material comprises polyacrylamide. In some embodiments, the polymer material comprises carrageenan. In some embodiments, the polymer material comprises gelatin. In some embodiments, the polymer material comprises polyethylene glycol (PEG). In some embodiments, the polymer material comprises polyethyleneglycol diacrylate (PEGDA). In some embodiments, the poly mer material comprises alginate. In some embodiments, the polymer material comprises polyethylene terephthalate. In some embodiments, the polymer material comprises cyclic olefin polymer. In some embodiments, the polymer material comprises cyclic olefin copolymer. In some embodiments, the polymer material comprises styrene-acrylonitrile. In some embodiments, the polymer material comprises polyvinyl butyral. In some embodiments, the polymer material comprises polyethylene naphthalate. In some embodiments, the polymer material comprises polyvinyl alcohol. In some embodiments, the polymer material comprises polylactic acid. In some embodiments, the polymer material comprises epoxy. In some embodiments, the polymer material comprises poly(allyl diglycol carbonate).

[0261] In some embodiments, the polymer material can be combined with one or more additional components that provide functionality to the polymer, where the additional component(s) can diffuse or otherwise be transmitted into the partitions of the partition matrix (e.g., in order to interact with targets captured in the partitions of the partition matrix). Embodiments of the additional component(s) can include one or more of: chemical probes, affinity ligands, enzymatic components, dyes (e.g., SYBR Green, hydrolysis probes, nonhydrolysis probes, cell viability-associated dyes, etc.), sequence-specific capture agents (e.g., antisense oligonucleotides, LNA-modified probes, PNA clamps, morpholino oligomers, etc.), CRISPR guide RNAs, molecular beacons, enzymes (e.g., polymerases, reverse transcriptases, ligases, nickases, recombinases, strand-displacing polymerases, etc ), small molecules (e.g., bis-intercalators (e.g., YOYO-1), minor-groove binders (e.g., netropsin, distamycin), G- quadruplex stabilizers (e.g., TMPyP4, BRACO-19), etc.), cell-penetrant viability dyes (calcein-AM, SYTO dyes), membrane-impermeant exclusion dyes (7-AAD, TO-PRO-3), lectins, aptamers, antibody-conjugated fluorophores, and metabolic reporters (resazurin, tetrazolium salts), metal-chelating probes, click-chemistry tags, barcoding oligos, lysis reagents, permeabilization reagents, fixing reagents, and / or other materials.

[0262] In some embodiments, the polymer material can be in a form of a solution (e.g., a polymer material-containing solution). In variations of Step S280, the polymer material can be combined with a solution to provide suitable osmolarity / osmotic balance with one or more components of the partition matrix, in order to satisfy clarity threshold requirementsAtty. DocketNo.: 43161-64728 / WO (003WO) described herein. In a specific example, the solution for providing osmolarity / osmotic balance includes components of the continuous phase described herein, which can include one or more of: a surfactant, such as a non-ionic, nondenaturing detergent that is miscible with water (e.g., octyl phenoxypoly ethoxy ethanol, another octylphenol surfactant, another phenol surfactant, etc.); water (e g., PCR-certified water), a sugar solution (e.g., D-(+)- Glucose solution); potassium chloride; magnesium chloride; tris-HCl; sodium azide; and other components. In variations, the solution can further include a pre-polymer component (e.g., agarose, UV -based pre-polymer, chemical crosslinking-based prepolymer, etc.) such that the polymer material can be actively transitioned to a set state (e.g., solidified or crosslinked state) with controlled application of a stimulus. In some embodiments, the polymer material (e.g.. polymer material-containing solution) comprises precursors (e.g., one or more monomer components) of polymer materials disclosed herein.

[0263] In some embodiments, the polymer material is in the form of a polymer materialcontaining solution. In some embodiments, the polymer material -containing solution comprises 0.02 to 10% w / v% of the polymer material. In some embodiments, the polymer material-containing solution comprises at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0. 1%, 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.1%, at least 1.2%. at least 1.3%, at least 1.4%, at least 1.5%, at least 1.6%. at least 1.7%, at least 1.8%, at least 1.9%, at least 2%. at least 2.1%, at least 2.2%, at least 2.3%, at least 2.4%, at least 2.5%, at least 2.6%, at least 2.7%, at least 2.8%, at least 2.9%, at least 3%, at least 3.1%, at least 3.2%, at least 3.3%, at least 3.4%, at least 3.5%, at least 3.6%, at least 3.7%, at least 3.8%, at least 3.9%, at least 4%, at least 4.1%, at least 4.2%, at least 4.3%, at least 4.4%, at least 4.5%, at least 4.6%. at least 4.7%, at least 4.8%, at least 4.9%, at least 5%, at least 5.1%, at least 5.2%, at least 5.3%, at least 5.4%, at least 5.5%, at least 5.6%, at least 5.7%, at least 5.8%, at least 5.9%, at least 6%, at least 6.1%, at least 6.2%, at least 6.3%, at least 6.4%, at least 6.5%, at least 6.6%, at least 6.7%, at least 6.8%, at least 6.9%, at least 7%, at least 7. 1%, at least 7.2%. at least 7.3%, at least 7.4%, at least 7.5%, at least 7.6%, at least 7.7%, at least 7.8%, at least 7.9%, at least 8%, at least 8.1%, at least 8.2%, at least 8.3%, at least 8.4%, at least 8.5%, at least 8.6%, at least 8.7%, at least 8.8%, at least 8.9%, at least 9%, at least 9.1%, at least 9.2%, at least 9.3%, at least 9.4%, at least 9.5%, at least 9.6%, at least 9.7%, at least 9.8%, at least 9.9%, at least 10%, or greater (in w / v%) of the polymer material. In some embodiments, the polymer material-containing solution comprises up to 0.02%, up to 0.03%, up to 0.04%, up to 0.05%, up to 0.06%, up to 0.07%, upAtty. DocketNo.: 43161-64728 / WO (003WO) to 0.08%, up to 0.09%, up to 0.1%, 0.2%, up to 0.3%, up to 0.4%, up to 0.5%, up to 0.6%, up to 0.7%, up to 0.8%, up to 0.9%. up to 1%, up to 1.1%, up to 1.2%. up to 1.3%, up to 1.4%, up to 1.5%, up to 1.6%, up to 1.7%, up to 1.8%, up to 1.9%, up to 2%, up to 2.1%, up to 2.2%, up to 2.3%, up to 2.4%, up to 2.5%, up to 2.6%, up to 2.7%, up to 2.8%, up to 2.9%, up to 3%, up to 3. 1%, up to 3.2%, up to 3.3%, up to 3.4%, up to 3.5%, up to 3.6%, up to 3.7%, up to 3.8%, up to 3.9%, up to 4%. up to 4.1%, up to 4.2%, up to 4.3%, up to 4.4%, up to 4.5%, up to 4.6%, up to 4.7%. up to 4.8%, up to 4.9%, up to 5%, up to 5. 1%, up to 5.2%, up to 5.3%, up to 5.4%, up to 5.5%, up to 5.6%, up to 5.7%, up to 5.8%, up to 5.9%, up to 6%, up to 6.1%, up to 6.2%, up to 6.3%, up to 6.4%, up to 6.5%, up to 6.6%, up to 6.7%, up to 6.8%, up to 6.9%, up to 7%, up to 7.1%, up to 7.2%, up to 7.3%, up to 7.4%, up to 7.5%, up to 7.6%, up to 7.7%, up to 7.8%, up to 7.9%. up to 8%, up to 8.1%, up to 8.2%, up to 8.3%, up to 8.4%, up to 8.5%, up to 8.6%, up to 8.7%, up to 8.8%, up to 8.9%, up to 9%, up to 9.1%, up to 9.2%, up to 9.3%, up to 9.4%, up to 9.5%, up to 9.6%, up to 9.7%, up to 9.8%, up to 9.9%, up to 10%, or greater (in w / v%) of the polymer material. In some embodiments, the polymer material-containing solution comprises from about 0.02 to about 0.1% w / v%, from about 0. 1 to about 0.25% w / v%, from about 0.25 to about 0.5% w / v%, from about 0.5 to about 0.75% w / v%, from about 0.75 to about 1% w / v%, from about 1 to about 1.25% w / v%, from about 1.25 to about 1.5% w / v%, from about 1.5 to about 1.75% w / v%, from about 1.75 to about 2% w / v%. from about 2 to about 2.25% w / v%, from about 2.25 to about 2.5% w / v%, from about 2.5 to about 2.75% w / v%, from about 2.75 to about 3% w / v%, from about 3 to about 3.5% w / v%, from about 3.5 to about 4% w / v%, from about 4 to about 4.5% w / v%, from about 4.5 to about 5% w / v%, from about 5 to about 5.5% w / v%, from about 5.5 to about 6% w / v%, from about 6 to about 6.5% w / v%, from about 6.5 to about 7% w / v%, from about 7 to about 7.5% w / v%. from about 7.5 to about 8% w / v%, from about 8 to about 8.5% w / v%, from about 8.5 to about 9% w / v%, from about 9 to about 9.5% w / v%, from about 9.5 to about 10% w / v%, or any intermediate range (in w / v%) of the polymer material. In some embodiments, the polymer material-containing solution comprises the polymer material (e.g., in an amount or concentration as disclosed herein) in 20 to 100% of a continuous phase described herein. In some embodiments, the polymer material-containing solution comprises 0.02 to 10% w / v% of the polymer material in 20 to 100% of the continuous phase described herein.

[0264] In specific examples, the polymer material-containing solution comprises 0.02 to 10% w / v% of agarose. In some embodiments, the polymer material-containing solution comprises at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, atAtty. DocketNo.: 43161-64728 / WO (003WO) least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%. at least 0.9%, at least 1%, at least 1.1%, at least 1.2%, at least 1.3%, at least 1.4%, at least 1.5%, at least 1.6%, at least 1.7%, at least 1.8%, at least 1.9%, at least 2%, at least 2.1%, at least 2.2%, at least 2.3%, at least 2.4%, at least 2.5%, at least 2.6%, at least 2.7%, at least 2.8%, at least 2.9%, at least 3%, at least 3.1%, at least 3.2%, at least 3.3%, at least 3.4%. at least 3.5%, at least 3.6%, at least 3.7%, at least 3.8%, at least 3.9%, at least 4%, at least 4. 1%, at least 4.2%, at least 4.3%, at least 4.4%, at least 4.5%, at least 4.6%, at least 4.7%, at least 4.8%, at least 4.9%, at least 5%, at least 5.1%, at least 5.2%, at least 5.3%, at least 5.4%, at least 5.5%, at least 5.6%, at least 5.7%, at least 5.8%, at least 5.9%, at least 6%, at least 6. 1%, at least 6.2%, at least 6.3%, at least 6.4%, at least 6.5%, at least 6.6%, at least 6.7%, at least 6.8%. at least 6.9%, at least 7%, at least 7.1%, at least 7.2%, at least 7.3%, at least 7.4%, at least 7.5%, at least 7.6%, at least 7.7%, at least 7.8%, at least 7.9%, at least 8%, at least 8.1%, at least 8.2%, at least 8.3%, at least 8.4%, at least 8.5%, at least 8.6%, at least 8.7%, at least 8.8%, at least 8.9%, at least 9%, at least 9.1%, at least 9.2%, at least 9.3%, at least 9.4%. at least 9.5%, at least 9.6%, at least 9.7%, at least 9.8%, at least 9.9%, at least 10%, or greater (in w / v%) of agarose. In some embodiments, the polymer material -containing solution comprises up to 0.02%, up to 0.03%, up to 0.04%, up to 0.05%, up to 0.06%, up to 0.07%, up to 0.08%, up to 0.09%, up to 0.1%, 0.2%, up to 0.3%, up to 0.4%, up to 0.5%. up to 0.6%, up to 0.7%, up to 0.8%, up to 0.9%, up to 1%, up to 1.1%, up to 1.2%, up to 1.3%. up to 1.4%, up to 1.5%, up to 1.6%. up to 1 .7%, up to 1.8%, up to 1.9%, up to 2%, up to 2. 1 %, up to 2.2%, up to 2.3%, up to 2.4%, up to 2.5%, up to 2.6%, up to 2.7%, up to 2.8%, up to 2.9%, up to 3%, up to 3.1%, up to 3.2%, up to 3.3%, up to 3.4%, up to 3.5%, up to 3.6%, up to 3.7%, up to 3.8%, up to 3.9%, up to 4%, up to 4.1%, up to 4.2%, up to 4.3%. up to 4.4%, up to 4.5%, up to 4.6%, up to 4.7%, up to 4.8%, up to 4.9%, up to 5%, up to 5.1%, up to 5.2%, up to 5.3%, up to 5.4%, up to 5.5%, up to 5.6%, up to 5.7%, up to 5.8%, up to 5.9%, up to 6%, up to 6.1%, up to 6.2%, up to 6.3%, up to 6.4%, up to 6.5%, up to 6.6%, up to 6.7%, up to 6.8%, up to 6.9%, up to 7%, up to 7.1%, up to 7.2%, up to 7.3%. up to 7.4%, up to 7.5%, up to 7.6%, up to 7.7%, up to 7.8%, up to 7.9%, up to 8%, up to 8. 1%, up to 8.2%, up to 8.3%, up to 8.4%, up to 8.5%, up to 8.6%, up to 8.7%, up to 8.8%, up to 8.9%, up to 9%, up to 9.1%, up to 9.2%, up to 9.3%, up to 9.4%, up to 9.5%, up to 9.6%, up to 9.7%, up to 9.8%, up to 9.9%, up to 10%, or greater (in w / v%) of agarose. In some embodiments, the polymer material-containing solution comprises from about 0.02 to about 0.1% w / v%, from about 0.1 to about 0.25% w / v%. from about 0.25 to about 0.5% w / v%, from about 0.5 to about 0.75% w / v%, from about 0.75 toAtty. DocketNo.: 43161-64728 / WO (003WO) about 1% w / v%, from about 1 to about 1.25% w / v%, from about 1.25 to about 1.5% w / v%, from about 1.5 to about 1.75% w / v%, from about 1.75 to about 2% w / v%, from about 2 to about 2.25% w / v%, from about 2.25 to about 2.5% w / v%, from about 2.5 to about 2.75% w / v%, from about 2.75 to about 3% w / v%, from about 3 to about 3.5% w / v%, from about 3.5 to about 4% w / v%, from about 4 to about 4.5% w / v%, from about 4.5 to about 5% w / v%, from about 5 to about 5.5% w / v%. from about 5.5 to about 6% w / v%, from about 6 to about 6.5% w / v%, from about 6.5 to about 7% w / v%, from about 7 to about 7.5% w / v%, from about 7.5 to about 8% w / v%, from about 8 to about 8.5% w / v%, from about 8.5 to about 9% w / v%, from about 9 to about 9.5% w / v%, from about 9.5 to about 10% w / v%, or any intermediate range (in w / v%) of agarose. In some embodiments, the polymer materialcontaining solution comprises agarose (e.g.. in an amount or concentration as disclosed herein) in 20 to 100% of a continuous phase described herein. In some embodiments, the polymer material -containing solution comprises 0.02 to 10% w / v% of agarose in 20 to 100% of the continuous phase described herein.

[0265] In specific examples, the polymer material (e.g., polymer material -containing solution) includes from 0.02 to 3% w / v% of an agarose solution in 20 to 100% of the continuous phase described herein. In some embodiments, the polymer material includes at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%. at least 0.7%, at least 0.8%, at least 0.9%, at least 1%. at least 1.1%, at least 1.2%, at least 1 .3%, at least 1 .4%, at least 1 .5%, at least 1 .6%, at least 1 .7%, at least 1.8%, at least 1.9%, at least 2%, at least 2.1%, at least 2.2%, at least 2.3%, at least 2.4%, at least 2.5%, at least 2.6%, at least 2.7%, at least 2.8%, at least 2.9%, at least 3%, or greater (in w / v%) of an agarose solution in 20 to 100% of the continuous phase described herein. In some embodiments, the polymer material includes up to 0.02%, up to 0.03%, up to 0.04%, up to 0.05%, up to 0.06%, up to 0.07%, up to 0.08%, up to 0.09%, up to 0.1%, 0.2%, up to 0.3%, up to 0.4%, up to 0.5%, up to 0.6%, up to 0.7%, up to 0.8%, up to 0.9%, up to 1%, up to 1.1%, up to 1.2%, up to 1.3%, up to 1.4%. up to 1.5%, up to 1.6%, up to 1.7%, up to 1.8%, up to 1.9%, up to 2%, up to 2. 1%, up to 2.2%, up to 2.3%, up to 2.4%, up to 2.5%, up to 2.6%, up to 2.7%, up to 2.8%, up to 2.9%, up to 3%, or greater (in w / v%) of an agarose solution in 20 to 100% of the continuous phase described herein. In some embodiments, the polymer material includes from about 0.02 to about 0.1% w / v%, from about 0.1 to about 0.25% w / v%, from about 0.25 to about 0.5% w / v%. from about 0.5 to about 0.75% w / v%, from about 0.75 to about 1% w / v%, from about 1 to about 1.25% w / v%, from about 1.25 toAtty. DocketNo.: 43161-64728 / WO (003WO) about 1.5% w / v%, from about 1.5 to about 1.75% w / v%, from about 1.75 to about 2% w / v%, from about 2 to about 2.25% w / v%, from about 2.25 to about 2.5% w / v%, from about 2.5 to about 2.75% w / v%, from about 2.75 to about 3% w / v%, or any intermediate range (in w / v%) of an agarose solution in 20 to 100% of the continuous phase described herein.

[0266] In relation to transmitting the polymer material in the flow state, the polymer material can be provided in the flow state by using heat, a chemical agent, a pH shift, a light stimulus, removal of a light stimulus, or another mechanism. In the specific examples, the polymer material is heated (e.g., microwaved, heated with a heating element, etc.) prior to transmission into the collecting container. Alternatively, the polymer material can be provided to the partition matrix in a set state and transitioned to the flow state (e.g., with heating, with another mechanism) prior to Step S290.

[0267] In relation to Steps S210 through S240 of method 200, Step S280 can include removing fluid layers (e.g., excess fluid layers) that do not form the resultant partition matrix, from the collecting container, prior to addition of the polymer material. In specific examples, removing excess fluid layers can include aspirating continuous phase and / or immiscible layer remnants from the collecting container prior to addition of the polymer material. Removal of excess fluid layers can be performed before or after transmission of heat (e.g., as in Step S230, with respect to thermocycling contents of the collecting container or otherwise heating or cooling the collecting container).

[0268] In a specific example, from 20 pL to 200 pL of the polymer material can be added to the collecting container over the partition matrix, prior to immobilization of the partition matrix. In some embodiments, up to 20 pL, up to 25 pL, up to 30 pL, up to 35 pL, up to 40 pL, up to 45 pL, up to 50 pL, up to 55 pL, up to 60 pL, up to 65 pL, up to 70 pL, up to 75 pL. up to 80 pL, up to 85 pL, up to 90 pL, up to 95 pL. up to 100 pL, up to 105 pL, up to 110 pL, up to 115 pL, up to 120 pL, up to 125 pL, up to 130 pL, up to 135 pL, up to 140 pL, up to 145 pL, up to 150 pL, up to 155 pL, up to 160 pL, up to 165 pL, up to 170 pL, up to 175 pL, up to 180 pL, up to 185 pL, up to 190 pL, up to 195 pL, or up to 200 pL of the polymer material can be added to the collecting container over the partition matrix, prior to immobilization of the partition matrix.

[0269] Step S290 recites: transitioning the polymer material to a set state within the collecting container, which functions to immobilize the partition matrix. Step S290 can also function to re-pack the partition matrix, such that the partitions are provided in a packed bulk configuration upon immobilization. Immobilization can be permanent or reversable, depending upon whether the set state is permanent or reversable. In variations, transitioningAtty. DocketNo.: 43161-64728 / WO (003WO) the polymer material to the set state can include one or more of: cooling the polymer material; using a chemical agent (e.g.. catalyst) to transition the polymer material to the set state; inducing a pH shift to transition the polymer material to the set state; applying an electromagnetic stimulus to transition the polymer material to the set state; or applying another stimulus to transition the polymer material to the set state. As such, in variations, transitioning the polymer material to the set state can include cross-linking the polymer material.

[0270] Step S290 can be performed in coordination with applying a force to contents of the collecting container, in order to re-pack the partition matrix. In variations, the force can be attributed to centrifugation, applied positive pressure, agitation (e.g., with a vortex), applied magnetic force, and or another suitable force.

[0271] In specific examples, Step S290 includes centrifuging the collecting container to repack the partition matrix after the polymer material has been provided in Step S280, while the polymer material cools and transitions from the flow state to the set state. As such, S290 can include transitioning the polymer material to the set state by centrifuging the collecting container in coordination with cooling of the polymer material within the collecting container.

[0272] In a specific example, the collecting container is centrifuged at 16000g for 25 minutes. However, in variations of the specific example spinning can be performed at 1,000g, 2,000g. 3,000g, 4,000g, 5,000g, 6.000g. 7,000g. 8,000g, 9,000g, 10,000g, 11,000g, 12.0000g, 13,000g, 14,000g, 15,000g, 16,000g, 17,000g, 18,000g, 19,000g, 20,000g, 30,000g, any intermediate value, or greater than 30,000g. Duration of spinning can be 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes. 9 minutes, 10 minutes, 11 minutes, 12 minutes. 13 minutes, 14 minutes, 15 minutes. 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, any intermediate value, or greater than 50 minutes. Spinning can be performed in an environment with environmental temperature control, in order to control the rate at which the polymer material cools (or is heated). In specific examples, the temperature of the centrifuge or other force-application environment can be maintained at a temperature above the transition temperature of the polymer material. In specific examples, the temperature of the centrifuge or other forceapplication environment can be maintained at the transition temperature of the polymer material. In specific examples, the temperature of the centrifuge or other force-application environment can be maintained at a temperature below the transition temperature of the polymer material. In specific examples, the temperature of the centrifuge or other force-Atty. DocketNo.: 43161-64728 / WO (003WO) application environment can be cycled around the transition temperature of the polymer material. In some embodiments, the transition temperature of the polymer material is a glass transition temperature of the polymer material. In some embodiments, the transition temperature of the polymer material is a melting temperature of the polymer material. In some embodiments, the transition temperature of the polymer material is at least 40 °C, at least 45 °C, at least 50 °C, at least 55 °C, at least 60 °C, at least 65 °C, at least 70 °C, at least 75 °C, at least 80 °C, at least 85 °C, at least 90 °C, at least 95 °C, at least 100 °C, at least 105 °C, at least 110 °C, at least 115 °C, at least 120 °C, at least 125 °C, at least 130 °C, at least 135 °C, at least 140 °C, at least 145 °C, at least 150 °C, any other intermediate value, or greater. In some embodiments, the transition temperature of the polymer material is 40 °C or lower, 45 °C or lower, 50 °C or lower. 55 °C or lower, 60 °C or lower, 65 °C or lower. 70 °C or lower, 75 °C or lower, 80 °C or lower, 85 °C or lower, 90 °C or lower, 95 °C or lower, 100 °C or lower, 105 °C or lower, 110 °C or lower, 115 °C or lower, 120 °C or lower, 125 °C or lower, 130 °C or lower, 135 °C or lower, 140 °C or lower, 145 °C or lower, 150 °C or lower, any other intermediate value, or lower.

[0273] After Step S290, clarity of the immobilized partition matrix is preferably unaffected, in relation to clarity of the partition matrix prior to immobilization. However, variations of the immobilized partition matrix may not have similar clarity7to the partition matrix prior to immobilization, in relation to other applications of use of an immobilized partition matrix. For instance, the immobilized partition matrix may not have suitable clarity7for interrogation according to methods described herein; however, upon reception at a test facility, the polymer material used to immobilize the partition matrix can be transitioned back to a flow state (e.g., w ith reversal of the set state), to return the partition matrix to a condition where clarity surpasses the clarity threshold.6.3.6.I. Methods - Implementation

[0274] As shown in FIG. 10, an embodiment of a method 300 for generation of partitions includes: providing an assembly S310 including: a first substrate defining one or more reservoirs, a membrane layer including a distribution of holes positioned downstream of the one or more reservoirs, one or more sealing bodies positioned adjacent to the membrane layer and including a set of openings aligned w ith the set of reservoirs, and optionally one or more fasteners configured to retain the assembly in position relative to one or more collecting containers containing a continuous phase and an immiscible layer over the continuous phase; optionally, receiving a sample fluid into the one or more reservoirs S320 (optionally, with aAtty. DocketNo.: 43161-64728 / WO (003WO) driving solution over the sample fluid), wherein the sample fluid is intended for use in partition formation and is immiscible with the immiscible layer; and applying force (e.g., centrifugation, pressurization, etc.) to contents of the reservoirs / assembly S330, thereby driving the sample fluid from the one or more reservoirs, through the membrane layer, and into the one or more collecting containers.

[0275] Embodiments of the method 300 function to reliably generate monodisperse partitions of a partition matrix for various applications (as described herein, and at rates described herein), such as digital amplification; capture of target material at cellular, subcellular, and molecular scales; sample analysis benefitting from partition generation; or other suitable applications. Embodiments of the method 300 can also function to generate monodisperse partitions using devices that are non-microfluidic, disposable, or reusable, in a cost-effective manner.

[0276] In specific applications, the method 300 can be used to generate partitions with applications in one or more of: emulsion generation (e.g., single emulsion generation, double emulsion generation), microparticle generation, liposome generation, hydrogel microparticle generation, nucleic acid amplification (e.g., by polymerase chain reaction (PCR) methods, by isothermal methods such as loop-mediated isothermal amplification (LAMP), by recombinase polymerase amplification (RPA), by multiple displacement amplification (MDA), by helicase dependent amplification (HD A), by strand displacement amplification (SDA), by nicking enzyme amplification (NEAR), by transcription mediated amplification (TMA), by digital helicase dependent amplification, by RNaseH mediated amplification, by whole genome amplification (WGA), by rolling circle amplification, etc.) on purified DNA, cDNA, RNA, or directly from lysate (e.g., blood lysate); fluorescent in situ hybridization (FISH) with fluorescently tagged nucleic acids (e.g., PNA, LNA, DNA, RNA. etc.) or an indirect in situ hybridization approach using DIG or biotin; by an in vitro transcription or translation assay (e.g., whereby a colorimetric or fluorescent reporter is used for detection); partition PCR applied to samples derived from single cells (e.g., prokary otes, eukary otes), organelles, viral particles, and exosomes; partition analysis of proteins (e.g., by proximity ligation assays, etc.); sequencing applications (e.g., single molecule sequencing applications); monitoring or detection of products (e g., proteins, chemicals) released from single cells (e.g., interleukin released from immune cells); monitoring cell survival and / or division for single cells; monitoring or detection of enzymatic reactions involving single cells; target material capture at cellular (e.g., mammalian cell, bacterial cell, pathogen, viral, etc.) and sub-cellular (e.g.. organelle, molecular, etc.) scales; enumeration of heterogeneous cell populations in a sample;Atty. DocketNo.: 43161-64728 / WO (003WO) enumeration of individual cells or viral particles (e.g., by encapsulating cells in partitions with species-specific antibodies coupled with enzymes that react with substrate components in the partition to produce signals, etc.); monitoring of viral infections of a single host cell; and other suitable applications.

[0277] The method 300 can be implemented by an embodiment, variation, or example of the system 100 described herein, or can alternatively be implemented by another suitable system.6.4. Computer Systems

[0278] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. FIG. 13 shows a computer system 1101 that is programmed or otherwise configured to, for example, generate a lurality of partitions within a collecting container at a predetermined rate or variation in poly dispersity, transmit heat to and from the plurality of partitions within the collecting container, perform an optical interrogation operation with the plurality of partitions within the collecting container, or perform one or more steps of methods 400, 600 described herein.

[0279] The computer system 1101 can regulate various aspects of analysis, calculation, and generation of the present disclosure, such as, for example, generating a plurality of partitions within a collecting container at a predetermined rate or variation in polydispersity, transmitting heat to and from the plurality of partitions within the collecting container, or performing an optical interrogation operation with the plurality of partitions within the collecting container. The computer system 1 101 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

[0280] The computer system 1101 includes a central processing unit (CPU, also ‘“processor ’ and “computer processor” herein) 1105, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 101 also includes memory7or memory location 1110 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 1115 (e.g., hard disk), communication interface 1120 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 1 125, such as cache, other memory, data storage and / or electronic display adapters. The memory 1110, storage unit 1115, interface 1120 and peripheral devices 1125 are in communication with the CPU 1105 through a communication bus (solid lines), such as a motherboard. The storage unit 1115 can be a data storage unit (or data repository) for storing data. The computer system 1101 can be operatively coupled to a computer networkAtty. DocketNo.: 43161-64728 / WO (003WO)(“network’') 1130 with the aid of the communication interface 1120. The network 1130 can be the Internet, an internet and / or extranet, or an intranet and / or extranet that is in communication with the Internet.

[0281] In some embodiments, the network 1130 is a telecommunication and / or data network. The network 1130 can include one or more computer servers, which can enable distributed computing, such as cloud computing. For example, one or more computer servers may enable cloud computing over the network 1130 (“the cloud”) to perform various aspects of analysis, calculation, and generation of the present disclosure, such as, for example, generating a plurality of partitions within a collecting container at a predetermined rate or variation in polydispersity. Such cloud computing may be provided by cloud computing platforms such as, for example, Amazon Web Services (AWS). Microsoft Azure, Google Cloud Platform, and IBM cloud. In some embodiments, the network 1130, with the aid of the computer system 1101, can implement a peer-to-peer network, which may enable devices coupled to the computer system 101 to behave as a client or a server.

[0282] The CPU 1105 may comprise one or more computer processors and / or one or more graphics processing units (GPUs). The CPU 1105 can execute a sequence of machine- readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory71110. The instructions can be directed to the CPU 1105, which can subsequently program or otherwise configure the CPU 1105 to implement methods of the present disclosure. Examples of operations performed by the CPU 1 105 can include fetch, decode, execute, and writeback.

[0283] The CPU 1105 can be part of a circuit, such as an integrated circuit. One or more other components of the system 1101 can be included in the circuit. In some embodiments, the circuit is an application specific integrated circuit (ASIC).

[0284] The storage unit 1115 can store files, such as drivers, libraries and saved programs. The storage unit 1115 can store user data, e.g., user preferences and user programs. In some embodiments, the computer system 1101 can include one or more additional data storage units that are external to the computer system 1101, such as located on a remote server that is in communication with the computer system 1101 through an intranet or the Internet.

[0285] The computer system 1101 can communicate with one or more remote computer systems through the network 1130. For instance, the computer system 1101 can communicate with a remote computer system of a user. Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad. Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device.Atty. DocketNo.: 43161-64728 / WO (003WO)Blackberry®), or personal digital assistants. The user can access the computer system 101 via the network 1130.

[0286] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 1101, such as, for example, on the memory 1110 or electronic storage unit 1115. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 1105. In some embodiments, the code can be retrieved from the storage unit 1115 and stored on the memory 11 10 for ready access by the processor 1105. In some situations, the electronic storage unit 1115 can be precluded, and machine-executable instructions are stored on memory 1110.

[0287] The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.

[0288] Embodiments of the systems and methods provided herein, such as the computer system 1101, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and / or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, or disk drives, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that cany such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible "storage” media, terms such as computerAtty. DocketNo.: 43161-64728 / WO (003WO) or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[0289] Hence, a machine readable medium, such as computer-executable code, may take many forms, including a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer- readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0290] The computer system 1 101 can include or be in communication with an electronic display 135 that comprises a user interface (UI) 1140 for providing, for example, a visual display indicative of generating a plurality of partitions within a collecting container at a predetermined rate or variation in polydispersily. transmitting heat to and from the plurality of partitions within the collecting container, performing an optical interrogation operation with the plurality of partitions within the collecting container, or of steps including performing an optical interrogation operation with a matrix. Examples of UIs include, without limitation, a graphical user interface (GUI) and web-based user interface.

[0291] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 1105. The algorithm can, for example, generate a plurality of partitions within a collecting container at a predetermined rate or variation in poly dispersity.Atty. DocketNo.: 43161-64728 / WO (003WO)6.5. Conclusions

[0292] The FIGs illustrate the architecture, functionality and operation of possible implementations of systems, methods and computer program products according to preferred embodiments, example configurations, and variations thereof. In this regard, each block in the flowchart or block diagrams may represent a module, segment, or, if applicable, portion of code, which comprises one or more executable instructions for implementing the specified logical function(s). It should also be noted that, in some alternative implementations, the functions noted in the block can occur out of the order noted in the FIGs. For example, two blocks shown in succession may, in fact, be executed substantially concurrently, or the blocks may sometimes be executed in the reverse order, depending upon the functionality involved. It will also be noted that each block of the block diagrams or flowchart illustration, and combinations of blocks in the block diagrams or flowchart illustration, can be implemented by special purpose hardware-based systems that perform the specified functions or acts, or combinations of special purpose hardware and computer instructions.

[0293] It should be understood from the foregoing that, while particular implementations have been illustrated and described, various modifications may be made thereto and are contemplated herein. It is also not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the preferable embodiments herein are not meant to be construed in a limiting sense. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. Various modifications in form and detail of the embodiments of the invention will be apparent to a person skilled in the art. It is therefore contemplated that the invention shall also cover any such modifications, variations and equivalents. It is intended that the following claims define the scope of the invention and that methods and structures w ithin the scope of these claims and their equivalents be covered thereby.Atty. DocketNo.: 43161-64728 / WO (003WO)7. EXAMPLES7.1. Example 1 - Calibration Samples

[0294] In specific examples, calibration samples for calibrating instruments used to scan samples were configured as distributions of dyed beads in a solidified polymer material, as follows:

[0295] Samples for calibrating uniformity7measurements for non-immobilized and / or immobilized samples: Exemplary compositions can include: < 10% agarose (e.g., by volume); < 50% Histodenz (e.g., by volume); > 40% DI water (e.g., by volume); < 1% ATTO488 / 532 / 594 / 647 free carboxylic acid (e.g., by volume).

[0296] Samples for calibrating thickness measurements for non-immobilized and / or immobilized samples: Exemplary7compositions can include: < 10% agarose (e.g., byvolume); < 50% Histodenz (e.g., by volume); > 40% DI water (e.g., by volume); < 1% ATTO488 / 532 / 594 / 647 free carboxylic acid (e.g., by volume); < 1% Spherotech™ submicron beads (e.g., Spherotech™ fluorescent purple, Spherotech™ fluorescent yellow, Spherotech™ fluorescent nile red, Spherotech™ fluorescent sky blue).

[0297] Samples for calibrating immobilized samples (e.g.. stored at 4 °C): Exemplary compositions can include: < 10% agarose (e.g., by volume); < 50% Histodenz (e.g., by volume); > 40% DI water (e g., by volume); and < 1 % Alexa488 / Atto532(NHS) / Alexa594 / Atto647(NHS) dyes (e.g., by volume).

Claims

Atty. DocketNo.: 43161-64728 / WO (003WO)CLAIMSWhat is claimed is:

1. A method comprising: fixing positions of a set of partitions of a partition matrix by immobilizing the partition matrix within a collecting container upon: transmitting a polymer material in a flow state into the collecting container and over the partition matrix, wherein the partition matrix has an optical clarity above a threshold level of clarity; and transitioning the polymer material to a set state within the collecting container, while maintaining optical clarity of the partition matrix above the threshold level of clarity.

2. The method of claim 1, wherein the partition matrix comprises a set of aqueous partitions generated from a sample, wherein the set of aqueous partitions is stabilized in position in a close-packed format within an aqueous continuous phase, and wherein each of the set of aqueous partitions comprises a thin film that is immiscible with the aqueous continuous phase.

3. The method of claim 1, wherein each partition of the set of partitions of the partition matrix is surrounded by the polymer material.

4. The method of claim 1, wherein the polymer material is provided at a surface of the partition matrix as a cap.

5. The method of claim 1, wherein the polymer material comprises a thermoplastic polymer material.

6. The method of claim 5, wherein the thermoplastic polymer material comprises agarose.

7. The method of claim 1, wherein the polymer material is in a form of a polymer material-containing solution.

8. The method of claim 7, wherein the poly mer material-containing solution comprises 0.02 to 3% w / v% of agarose.Atty. DocketNo.: 43161-64728 / WO (003WO)9. The method of claim 1, wherein the polymer material comprises a thermosetting polymer material.

10. The method of claim 1, wherein transmitting the polymer material in the flow state comprises heating the polymer matenal prior to transmission into the collecting container.

11. The method of claim 1 , wherein transitioning the polymer material to the set state comprises centrifuging the collecting container in coordination with cooling of the polymer material within the collecting container.

12. The method of claim 1, wherein transitioning the polymer material to the set state comprises cross-linking the polymer material.

13. The method of claim 1, wherein the set of partitions of the partition matrix contains a set of targets.

14. The method of claim 13, wherein each of the set of partitions contains at most one target of the set of targets.

15. The method of claim 13, wherein the set of targets comprises a set of nucleic acid targets.

16. The method of claim 13, wherein the set of targets comprises a set of protein targets.

17. The method of claim 1, wherein the partition matrix with the polymer material has optical clarity above the threshold level of clarity without use of refractive index matching between the partition matrix and the polymer material.

18. The method of claim 1, wherein the threshold level is at least 80% transmissivity of light.

19. A method for generating an immobilized partition matrix within a collecting container, the method comprising: generating a plurality of partitions within a collecting container at a rate of at least 1 million partitions / minute, each of the plurality' of partitions including an aqueous mixture for a digital analysis;Atty. DocketNo.: 43161-64728 / WO (003WO) stabilizing the plurality of partitions as a partition matrix in a close-packed format within a continuous phase, within a region of the collecting container; transmitting a polymer material in a flow state into the collecting container and over the partition matrix; and transitioning the polymer material to a set state within the collecting container, wherein the partition matrix with the polymer material has optical clarity above a threshold level of clarity without use of refractive index matching between the partition matrix and the polymer material, wherein the threshold level is at least 80% transmissivity of light, and wherein each partition of the plurality of partitions contains at most one target of a set of targets.

20. A system comprising: a partition matrix within a closed container, wherein a set of partitions of the partition matrix is immobilized within the closed container with a polymer material, and wherein the partition matrix with the polymer material has a level of optical clarity above a threshold level of clarity.

21. A method comprising : fixing positions of a set of targets of a sample within a matrix in three dimensions, wherein the matrix has a level of optical clarity greater than a threshold level, wherein fixing positions comprises: combining the sample with a set of processing reagents for a reaction, and a gelling material at a temperature above a melting temperature of the gelling material, and reducing the temperature of the sample, the set of processing reagents, and the gelling material below the melting temperature, thereby generating the matrix; linking amplified targets of the set of targets with a set of probes of the set of processing reagents upon performing the reaction; detecting signals emitted from probes of the set of probes associated with targets of the set of targets upon scanning the matrix with an optical detection system; and generating a characterization of the set of targets of the sample from the detected signals.Atty. DocketNo.: 43161-64728 / WO (003WO)22. The method of claim 21, wherein the set of targets comprises a set of nucleic acid targets.

23. The method of claim 21, wherein the gelling material comprises at least one of agarose, carrageenan, polyethyleneglycol diacrylate (PEGDA), and polyacrylamide.

24. The method of claim 21. wherein the set of processing reagents comprises a set of probes configured to associate with targets of the set of targets upon performing the reaction and to emit fluorescent signals upon associating with targets of the set of targets.

25. The method of claim 21, further comprising crosslinking the gelling material prior to performing the reaction.

26. The method of claim 21, wherein performing the reaction comprises performing the reaction below the melting temperature of the gelling material.

27. The method of claim 21, wherein the reaction comprises: an isothermal amplification reaction; or a rolling circle amplification reaction that physically links amplicons of a target template to the target template.

28. The method of claim 21, further comprising restricting movement of amplicons of a target template away from the target template.

29. The method of claim 28, wherein restricting movement comprises coupling amplicons of the target template to a region of the matrix in proximity to the target template.

30. The method of claim 28, wherein restricting movement comprises confining amplicons of the target template within a membrane.

31. The method of claim 21, wherein generating the characterization comprises one or more of: generating a count of the set of targets; determining co-occurrence of markers associated with each of a set of different analyte types; characterizing changes in detected signals over a set of time points; andAtty. DocketNo.: 43161-64728 / WO (003WO) characterizing changes in detected signals in response to a stimulus.

32. The method of claim 21, wherein the threshold level is at least 80% transmissivity7of light.

33. A method comprising: generating a count of a set of targets stabilized in position in three dimensions within a matrix having a level of optical clarity greater than a threshold level, wherein the threshold level is at least 80% transmissivity7of light, and wherein the set of targets comprises greater than 1000 targets.

34. The method of claim 33, wherein the matrix comprises a gelling material at a temperature below a melting temperature of the gelling material, and wherein generating the count comprises: linking a set of probes with amplicons of the set of targets upon performing a reaction below the melting temperature within the matrix; and detecting fluorescent signals emitted from the set of probes upon scanning a set of cross sections through the matrix with a light sheet system.

35. A system comprising: a matrix within a closed container, the matrix comprising a set of processing reagents for a reaction, and a set of targets stabilized in position in three dimensions within the matrix, wherein the matrix comprises a gelling material at a temperature below a melting temperature of the gelling material, wherein the reaction is configured to be performed below the melting temperature of the gelling material, and wherein the matrix comprises a level of optical clarity7greater than a threshold level.

36. The system of claim 35, wherein the set of processing reagents comprises a set of probes configured to associate with amplicons of the set of targets upon performing the reaction and to emit fluorescent signals upon associating with amplicons of the set of targets.

37. The system of claim 35, further comprising a light sheet system.Atty. DocketNo.: 43161-64728 / WO (003WO)38. The system of claim 35, wherein the gelling material comprises at least one of agarose, carrageenan, polyethyleneglycol diacrylate (PEGDA), and polyacrylamide.

39. The system of claim 35, wherein the set of targets comprises a set of nucleic acid targets, a set of protein targets, or a set of single cells.

40. The system of claim 35, wherein the threshold level is at least 80% transmissivity of light.

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