Method for assessing viability of microbial strains

Abstract

Described herein are methods for detecting viable cells in a sample.

Description

Attorney Docket No.: 273089 / KBS-012WO / 577219METHOD FOR ASSESSING VIABILITY OF MICROBIAL STRAINSCLAIM OF PRIORITY

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 720,519, filed November 14, 2024, which is incorporated by reference in its entirety.TECHNICAL FIELD

[0002] This invention relates to methods of detecting viable cells in a sample.BACKGROUND

[0003] Traditionally, the assessment of viability in a microbial consortium sample has been limited to a collective evaluation of all strains present, without the capability to differentiate and measure the viability of each strain individually. This limitation poses significant challenges in understanding the health and viability of specific strains within a consortium, which is crucial for the development and optimization of robust product formulations. Accordingly, there is a critical need in the field of live biotherapeutics and microbiome-based therapies to develop methods for assessing viability of strains in a microbial consortium.SUMMARY

[0004] In one aspect, the present disclosure provides a method of detecting viable cells in a sample, the method comprising:(a) staining the cells with a fixable viability dye;(b) washing away the fixable viability dye;(c) fixing the cells of the sample on an array;(d) performing an identifying assay on the cells;(e) adding a nuclear dye to the cells; and(f) mounting and imaging the cells with a microscope to assess the viability of the cells.

[0005] In some embodiments, the imaging with a microscope can produce a first, second, and third detectable signal,1581226294Attorney Docket No.: 273089 / KBS-012WO / 577219wherein the first detectable signal derives from the viability dye and is indicative of the presence of dead cells;wherein the second detectable signal derives from the nuclear dye and is indicative of the presence of a viable cell fraction; andwherein the third detectable signal derives from the identifying assay and is indicative of a known microbial strain.

[0006] In some embodiments, the identifying assay can include:(A) providing a first set of probes,wherein the first set of probes comprises at least one first encoding probe and at least one first emissive readout probe; and(B) contacting the first set of probes with the fixed cells to form a first complex; wherein the at least one first emissive readout probe emits a first color, and wherein the first color is assigned to a known microbial strain.

[0007] In some embodiments, the method further can include (e.l) incorporating controls into the sample prior to imaging, wherein the controls comprise community-based controls or strainspecific controls.

[0008] In some embodiments, the community -based control can include a preparation of “live” and “dead” controls, wherein the “live” control comprises a portion of the sample that is fixed and washed and has not been exposed to a viability dye; and wherein the “dead” control comprises a portion of the sample that is fixed and washed and has been exposed to a viability dye.

[0009] In some embodiments, the strain-specific control can include a preparation of “live” and “dead” pure culture of a known, individual strain, wherein the “live” pure culture comprises a portion of a culture of a known, individual strain that is fixed and washed and has not been exposed to a viability dye; and wherein the “dead” pure culture comprises a portion of a culture of a known, individual strain that is fixed and washed and has been exposed to a viability dye.

[0010] In some embodiments, the method further can include (e.2) performing a second identifying assay, wherein the second identifying assay produces a fourth detectable signal.

[0011] In some embodiments, the fourth detectable signal derives from the second identifying assay and is indicative of a known gene, a known protein, or a known marker of cell stress or cell viability.

[0012] In some embodiments, the second identifying assay can be indicative of a known gene,2581226294Attorney Docket No.: 273089 / KBS-012WO / 577219and wherein the second identifying assay comprises:contacting at least one second encoding probe with the cell to produce a second complex, wherein each encoding probe comprises an mRNA targeting sequence and an initiator sequence;adding two different DNA amplifier sequences to the second complex to produce a third complex, wherein each DNA amplifier comprises an initiator complimentary sequence and a readout sequence; andadding two second emissive readout probes to the third complex, wherein each second emissive readout probe comprises a fluorophore and a complimentary sequence to the second readout sequence of a corresponding DNA amplifier sequence.

[0013] In some embodiments, the second identifying assay can be indicative of a known protein, and wherein the second identifying assay comprises: identifying the presence of immunoglobulins on the surface of the cell, wherein the identifying the presence of immunoglobulins on the surface of a cell comprises incorporating an oligonucleotide-conjugated antibody or a fluorophore conjugated antibody.

[0014] In some embodiments, the method further can include (g) assessing the morphology of the cells.

[0015] In some embodiments, the morphology assessment can include assessing the area, volume, specific radius, Euler number, solidity, eccentricity, perimeter, convex area, orientation relative to neighbors, geometry, or shape of the cell.

[0016] Other aspects, embodiments, and features as disclosed herein will be apparent from the following description, the drawings, and the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 shows measurements and growth implications of a strain in the phylum Bacillota. On the left, the Bacillota strain was isolated at two points in its growth - stationary (black) and exponential (gray). In the middle, the normalized rRNA intensity is shown for the two states, as are the micrographs of fluorescent bacteria. Following freezing and then thawing, bacteria were re-cultured and monitored for growth. The sample isolated in the exponential phase reached saturation over 8 hours before those from the stationary phase when the same number of cells was added to the culture medium.

[0018] Figure 2 shows measurements and growth implications of a strain in the phylum3581226294Attorney Docket No.: 273089 / KBS-012WO / 577219Bacteroidota. On the left, the Bacteroidota strain was isolated at two points in its growth -stationary (black) and exponential (gray). In the middle, the normalized rRNA intensity is shown for the two states, as are the micrographs of fluorescent bacteria. Following freezing and then thawing, bacteria were re-cultured and monitored for growth. The sample isolated in the exponential phase reached saturation at a similar time as those from the stationary phase when the same number of cells was added to the culture medium.

[0019] Figure 3 shows, a mixed community of live and dead Escherichia coh and Enterococcus faecalis are shown. E. coli are identified with the pink color, and E. faecalis are identified with the green color, based on HiPR-FISH. Using a viability dye, dead cells can be removed from classification measurements (right).

[0020] Figure 4 shows classified microbiota in a complex synthetic community (on the left), following the application of viability measurements, dead microbiota were removed from classification (on the right).

[0021] Figure 5 shows the frequency of live and dead cells are enumerated for several strains shown in Figure 4.

[0022] Figure 6A shows the viability state of each segmented object in a canine stool sample (green is alive, red is dead), the raw signal intensity of microbiota is shown to the right (comprising rRNA signal, viability signal, and nuclear stain).

[0023] Figure 6B shows the viability ratio (higher ratio means more viability dye, which indicates the cell is more permeable and, therefore, likely to be dead) compared to the ribosomal RNA intensity for microbiota in canine stool.

[0024] Figure 7 shows lyophilized complex community was resuspended in media, and immediately exposed to heat (60°C) or placed in a 50 mL tube and rotated under atmospheric conditions. Cells were then stained with viability dye and assayed to determine rRNA (biomarker) intensity.

[0025] Figure 8 shows the normalized rRNA intensity is shown for the two states, as are the micrographs of fluorescent bacteria exposed to heat (60°C) or placed in a 50 mL tube and rotated under atmospheric conditions.INCORPORATION BY REFERENCE

[0026] All publications, patents, and patent applications mentioned in this specification are4581226294Attorney Docket No.: 273089 / KBS-012WO / 577219herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0027] In particular, the entire contents of each of the following patent applications are incorporated herein by reference in their entireties: PCT Application Nos.: PCT / US2019 / 021088; PCT / US2022 / 080355; and PCT / US2023 / 062917; and U.S. Application Nos.: 16 / 978,891; 18 / 058,171; and 18 / 171,850.DETAILED DESCRIPTION

[0028] It is to be appreciated that certain aspects, modes, embodiments, variations, and features of the present methods and compositions are described below in various levels of detail in order to provide a substantial understanding of the present disclosure.

[0029] Definitions

[0030] Where values are described as ranges, endpoints are included. Furthermore, it will be understood that such disclosure includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated.

[0031] “5’ -end” and “3 ’-end” refers to the directionality, e.g., the end-to-end orientation of a nucleotide polymer (e.g., DNA). The 5’-end of a polynucleotide is the end of the polynucleotide that has the fifth carbon.

[0032] The term “about,” as used herein, refers to + / - 10% of a recited value.

[0033] “Complementary” refers to the topological compatibility or matching together of interacting surfaces of two nucleotides as understood by those of skill in the art. Thus, two sequences are “complementary” to one another if they are capable of hybridizing to one another to form a stable anti-parallel, double-stranded nucleic acid structure. A first nucleotide is complementary to a second nucleotide if the nucleotide sequence of the first nucleotide is substantially identical to the nucleotide sequence of the nucleotide binding partner of the second nucleotide, or if the first nucleotide can hybridize to the second nucleotide under stringent hybridization conditions. Thus, the nucleotide whose sequence is 5'-TATAC-3' is complementary to a nucleotide whose sequence is 5'-GTATA-3'.

[0034] ‘Nucleotides,” “Nucleic acids,” “polynucleotide” or “oligonucleotide” refer to a polymeric-form of DNA and / or RNA (e.g., ribonucleotides, deoxyribonucleotides, or analogs5581226294Attorney Docket No.: 273089 / KBS-012WO / 577219thereof) of any length; e.g., a sequence of two or more ribonucleotides or deoxyribonucleotides. As used herein, the term “nucleotides” includes double- and single-stranded DNA, as well as double- and single-stranded RNA; it also includes modified and unmodified forms of a nucleotide (modifications to and of a nucleotide, for example, can include methylation, phosphorylation, and / or capping). In some embodiments, a nucleotide can be one of the following: a gene or gene fragment; genomic DNA; genomic DNA fragment; exon; intron; messenger RNA (mRNA); transfer RNA (tRNA); ribosomal RNA (rRNA); ribozyme; cDNA; recombinant nucleotide; branched nucleotide; plasmid; vector; isolated DNA of any sequence; isolated RNA of any sequence; any DNA described herein, any RNA described herein, primer or amplified copy of any of the foregoing.

[0035] In some embodiments, nucleotides can have any three-dimensional structure and may perform any function, known or unknown. The structure of nucleotides can also be referenced to by their 5’- or 3’- end or terminus, which indicates the directionality of the nucleotide sequence. Adjacent nucleotides in a single-strand of nucleotides are typically joined by a phosphodiester bond between their 3’ and 5’ carbons. However, different intemucleotide linkages could also be used, such as linkages that include a methylene, phosphoramidate linkages, etc. This means that the respective 5’ and 3’ carbons can be exposed at either end of the nucleotide sequence, which may be called the 5’ and 3’ ends or termini. The 5’ and 3’ ends can also be called the phosphoryl (PO4) and hydroxyl (OH) ends, respectively, because of the chemical groups attached to those ends. The term “nucleotides” also refers to both double- and single-stranded molecules.

[0036] In some embodiments, nucleotides can include modified nucleotides, such as methylated nucleotides and nucleotide analogs (including nucleotides with non-natural bases, nucleotides with modified natural bases such as aza- or deaza-purines, etc.). If present, modifications to the nucleotide structure can be imparted before or after assembly of the nucleotide sequence.

[0037] In some embodiments, the sequence of nucleotides can be interrupted by nonnucleotide components. One or more ends of the nucleotides can be protected or otherwise modified to prevent that end from interacting in a particular way (e.g. forming a covalent bond) with other nucleotides.

[0038] In some embodiments, nucleotides can be composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T). Uracil (U) can also be6581226294Attorney Docket No.: 273089 / KBS-012WO / 577219present, for example, as a natural replacement for thymine when the nucleotide is RNA. Uracil can also be used in DNA. Thus, the term “sequence” refers to the alphabetical representation of nucleotides or any nucleic acid molecule, including natural and non-natural bases.

[0039] When used in terms of length, for example 20 nt, “nt” refers to nucleotide(s).

[0040] As used herein a “taxon” refers to a group of one or more populations of an organism or organisms. In some embodiments, a “taxon” refers to a phylum, a class, an order, a family, a genus, a species, or a strain. In some embodiments, the disclosure includes providing a list of taxa of microorganisms. In some embodiments, the list of taxa of microorganisms is selected from a list of phyla, a list of classes, a list of orders, a list of families, a list of genera, or a list of species of microorganisms.

[0041] In analysis of a sample, a species can be a target of interest. For example, a species can include a taxonomic species.

[0042] A “pathogen” refers to any agent capable of causing an infection or a disease in a host, i.e., a cell or subject. In some embodiments, a pathogen is a microorganism or microbial fragment. In some embodiments, the pathogen may comprise a whole (infectious) pathogen cell, or a part of the pathogen cell, such as, a cell wall component of a microorganism. In some embodiments, the pathogen comprises a pathogen fragment, a pathogen debris, a pathogen nucleic acid, a pathogen lipoprotein, a pathogen surface glycoprotein, a pathogen membrane component, or a component released from the pathogen. In some embodiments, the pathogen is, is derived from, or is isolated from bacteria, fungus, prokaryote, virus, phage, or a misfolded protein (e.g., a prion). In some embodiments, the pathogen is genetically modified.

[0043] A “microbial strain” refers to any strain derived from a microbial species. A “microbial strain” may also be any strain that can potentially ameliorate a disease phenotype. In some embodiments, a microbial strain is any strain derived from a microbe and which does not cause a disease in a host, i.e., a cell or subject. In some embodiments, a microbial strain produces a therapeutic effect. In some embodiments, a microbial strain is a strain that acts against the pathogen. A microbial strain used in the methods described herein may derive from a healthy cell or subject and / or may contain healthy or desirable microbes. In some embodiments, a microbial strain is selected from the group consisting of a bacterium, a synthetic bacterium, a synthetic organism, a fungus, a virus, an archaea, a parasite, and a genetically modified organism. In some embodiments, a genetically modified organism is an organism wherein specific genes have been7581226294Attorney Docket No.: 273089 / KBS-012WO / 577219added, deleted, mutated, driven to high expressions, and / or suppressed to low expression levels. In some embodiments, the genetic modifications in the genetically modified organism are performed via electroporation, conjugation, transformation, transduction, CRISPR-Cas9 system, TALENs (Transcription Activator-Like Effector Nucleases), homologous recombination, sitespecific recombinases, or bacterial artificial chromosomes. In some embodiments, a microbial strain is abacterial strain such as a probiotic. The microbial strain of the methods described herein is to provide therapeutic / beneficial effects to the host and therefore does not cause a disease in said host. It is possible that microbial strains may be (or be derived from) strains that do not cause a disease in a normal host, but can cause disease in a subpopulation (e.g., an immunocompromised subject).

[0044] In the event of any term having an inconsistent definition between this application and a referenced document, the term is to be interpreted as defined herein.HiPR-Vie

[0045] Traditionally, the assessment of viability in a microbial consortium sample has been limited to a collective evaluation of all strains present, without the capability to differentiate and measure the viability of each strain individually. This limitation poses significant challenges in understanding the health and viability of specific strains within a consortium, which is crucial for the development and optimization of robust product formulations. The presently disclosed HiPR-Vie assay addresses this gap by enabling the precise assessment of the viability of individual strains within a consortium sample. This capability is essential for several reasons:

[0046] Enhanced process development: By evaluating the viability of each strain independently, there can be a better understanding on how different strains respond to various bioprocess conditions. This allows for more informed decisions during process development, leading to optimized growth and harvest conditions, and ultimately improving the overall viability and function of microbial consortium.

[0047] Improved formulation robustness: In live biotherapeutics consortium drug product, the health of each strain within a formulation is critical for ensuring the efficacy and stability of the final product. The methods disclosed herein enable the detection of strain’s health based on ribosomal RNA and viability, allowing for targeted strategies to enhance their health. This leads to more consistent and reliable formulations, which are essential for therapeutic success.8581226294Attorney Docket No.: 273089 / KBS-012WO / 577219

[0048] Tailored experimental design: Understanding the viability and health of individual strains now allows us to design more targeted experiments aimed at improving specific strains' health. This targeted approach not only accelerates the development process but also increases the likelihood of achieving robust, high-performing microbial formulations for drug products.

[0049] In summary, the methods disclose herein represent a significant advancement in the field, providing a powerful tool for improving process development and formulation robustness in consortium samples. The ability to assess individual strain health opens new avenues for optimizing live biotherapeutic products, ultimately contributing to more effective and consistent therapies.

[0050] Assessments of viability have been used in the past to optimize process development:

[0051] Live-dead staining + detection: Cells are stained with a dye that illuminates cells in one color if they are alive and another if they are dead (or alternatively, illuminates them only if they’re alive or dead). The cells are then measured on a microscope or with a flow cell to determine the number of alive cells and number of dead cells. A viability fraction can then be determined by calculating the fraction of alive cells over total cells. Current methods are generally not able to distinguish between taxon identity. Counting can be difficult by manual or automated means in these settings.

[0052] CFU counting: A stock can be diluted and streaked out onto plates and placed in proper conditions for growth. The number of colonies generated from a known volume and dilution can then be used to calculate the number of colony forming units per unit volume. In this case, the viable fraction of cells is not determined and again, taxonomy is unknown.

[0053] PCR based methods: Assays like propidium monoazide (PMA)-quantitative polymerase chain reaction can be used to detect viable cells in a sample. PMA selectively penetrates damaged cells and binds to DNA, this prevents PCR of the DNA to which it is bound. Specific PCR probes can be added to samples, if PCR products are generated, it suggests PMA is not bound and a cell is viable. While PCR probes can specify identity, each identification needs to be done as a separate isolate, and the ability to determine individual cells is not possible.

[0054] Accordingly, there is a critical need in the field of live biotherapeutics and microbiomebased therapies to develop methods for assessing viability of strains in a microbial consortium. The methods described in the present disclosure provide the ability to determine the viability and identity of microbes within complex communities. These methods result in various improvements9581226294Attorney Docket No.: 273089 / KBS-012WO / 577219such as:

[0055] 1. Quantification of rRNA intensity to assess microbial metabolic activity, potential for regrowth, or engraftment within complex environments.

[0056] 2. Measurement of DNA content, such as with DAPI or other probes, to evaluate replication potential of microbes in these communities.

[0057] 3. Spatial mapping of microbial interactions, including identification of microbes growing in close proximity or attached to one another (e.g., clumps) that occur during process development, or culturing, or that occur naturally (e.g. in fecal pellets).

[0058] 4. Assessment of differences in viability and rRNA intensity between microbes involved in these physical associations.

[0059] 5. Applications of the assays to the field of LBP drug product development, bioengineering, and others.

[0060] Further, HiPR-Vie offers a sophisticated approach to strain isolation by utilizing targeted probe panels to determine the viability of specific strains in a complex microbial community. By providing insights into which strains are alive or dead, researchers can make informed decisions on how to refine their isolation strategies. For instance, if a targeted strain of interest is found to be over 99% non-viable, attempting to isolate it may be inefficient, and the strategy can be adjusted to focus on strains with better viability.

[0061] Furthermore, HiPR-Vie can play a significant role in culture and media optimization. It enables simultaneous testing of multiple culture conditions, such as different media types, nutrients, and supplements, to determine the optimal environment that maximizes strain viability. By comparing the viable fraction across different conditions, researchers can efficiently identify the most favorable conditions for growth and isolation, accelerating the development of viable cultures for further study.

[0062] Ensuring the quality of LBPs or FMT material is crucial for therapeutic efficacy and patient safety. HiPR-Vie’ s per-taxon viability assessment can be extended beyond the development phase to serve as a vital quality control tool for final products. By evaluating the viability of each strain in a batch, HiPR-Vie helps determine whether the production process has maintained the intended microbial composition and viability standards.

[0063] HiPR-Vie is a powerful tool for advancing biological discovery, particularly in the context of host-microbe interactions. When applied to in vivo tissue sections immediately after10581226294Attorney Docket No.: 273089 / KBS-012WO / 577219animal sacrifice, HiPR-Vie can reveal which microbial strains are viable and their precise spatial locations within the tissue. This spatial mapping capability is crucial for understanding how specific strains interact with host cells and how they might contribute to health or disease.

[0064] Methods

[0065] The methods for detecting viable cells in a sample can be summarized as follows:(a) Sample is stained with a viability dye. Cells are stained for 30 minutes under amenable conditions (e.g. in anaerobic chamber). These dyes act by binding to amines in cells where the membrane is permeable.(b) The viability dye is washed away. Cells are fixed and washed.(c) A HiPR-FISH assay is performed to label taxa of interest in the community.(d) DAPI is added to the community at a concentration of 1-1000 ng / mL for 1-10 minutes.(e) The sample is mounted.(f) The sample is imaged on a hyperspectral microscope and data is processed using standard pipeline.

[0066] High Phylogenetic Resolution microbiome mapping by Fluorescence in situ Hybridization (HiPR-FISH), developed by the Applicant, is a versatile technology that uses binary encoding, spectral imaging, and machine learning based decoding to create micron-scale maps of the locations and identities of hundreds of microbial species in complex communities. See, for example, Shi, H. et al. “Highly multiplexed spatial mapping of microbial communities.” Nature vol. 588, 7839 (2020): 676-681, PCT Patent Publication WO 2019 / 173555, filed March 7, 2019; PCT Patent Application No. PCT / US2022 / 080355, filed on November 24, 2022, and U.S. Application No. 18 / 058,171, filed on November 24, 2022. The contents of the aforementioned disclosures are each incorporated herein by reference in their entireties.

[0067] HiPR-FISH has been able to distinguish between 1023 unique targets in a single experiment. It has been applied to numerous specimen types including mammalian tissue, food products, biofilms, and cultured epithelial cells with adherent bacteria. The present disclosure provides methods to incorporate this technology for identifying and isolating microbial strains from samples of interest.

[0068] Accordingly, the present disclosure provides a method of detecting viable cells in a sample, the method comprising:11581226294Attorney Docket No.: 273089 / KBS-012WO / 577219(a) staining the cells with a fixable viability dye;(b) washing away the fixable viability dye;(c) fixing the cells of the sample on an array;(d) performing an identifying assay on the cells;(e) adding a nuclear dye to the cells; and(f) mounting and imaging the cells with a microscope to assess the viability of the cells.

[0069] In some embodiments, the imaging with a microscope produces a first, second, and third detectable signal,wherein the first detectable signal derives from the viability dye and is indicative of the presence of dead cells;wherein the second detectable signal derives from the nuclear dye and is indicative of the presence of a viable cell fraction; andwherein the third detectable signal derives from the identifying assay and is indicative of a known microbial strain.

[0070] In some embodiments, the identifying assay comprises:(A) providing a first set of probes,wherein the first set of probes comprises at least one first encoding probe and at least one first emissive readout probe; and(B) contacting the first set of probes with the fixed cells to form a first complex; wherein the at least one first emissive readout probe emits a first color, and wherein the first color is assigned to a known microbial strain.

[0071] In some embodiments, the imaging acquires one emission spectra from the at least one first emissive readout probe.

[0072] In some embodiments, the viability of cells is assessed via a relative signal analysis or a barcode classification.

[0073] Since the viability dye stains any cell that is permeable and dead, detecting the signal is crucial for assessing viability. When using fixable dyes, there are two approaches for analyzing viability.

[0074] Relative Signal Analysis: Assess the amount of viability dye signal relative to a nuclear stain, such as DAPI. The viability dye signal — measured either by peak intensity or total12581226294Attorney Docket No.: 273089 / KBS-012WO / 577219integrated intensity — can be used to determine the viability status of the cells.

[0075] In some embodiments, the relative signal analysis measures the ratio between the amount of viability dye signal relative to the amount of nuclear dye signal.

[0076] Barcode Classification: Treat the viability dye as a unique “bit” in a barcode system, where dead cells register this bit as “1” and live cells as “0.” The first digit of the cell barcode is reserved for viability (e.g., X101, where “101” represents taxonomic identification, such as E. coli). In this system, “0101” represents a live E. coll, while “1101” represents a dead E. coli.

[0077] In some embodiments, the barcode classification assigns a bit to the viability dye, wherein the bit “1” is assigned to dead cells and bit “0” is assigned to live cells.

[0078] In some embodiments, wherein the method further comprises (e.l) incorporating controls into the sample prior to imaging, wherein the controls comprise community-based controls or strain-specific controls.

[0079] The detection of dead cells in the present methods is based on cell wall permeability, which varies across different taxa, leading to differential absorption of viability dyes (also with DAPI). To account for this variability, controls are with each experimental condition to distinguish live from dead cells. Two strategies for incorporating controls are used:

[0080] Community-Based Controls: For each microbial community, a “live” and “dead” control is created by dividing the initial sample into three parts at the beginning of the assay. One part serves as the test sample. The other two parts are immediately fixed and washed — one without exposure to viability dye (serving as a “live” control), and the other stained with viability dye (representing a “dead” control). Since all cells in these controls are permeabilized during fixation, the viability dye stains only the dead cells. All three samples undergo image processing, and a viability measurement is performed for each taxon to assess accuracy.

[0081] In some embodiments, the community-based control comprises a preparation of “live” and “dead” controls, wherein the “live” control comprises a portion of the sample that is fixed and washed and has not been exposed to a viability dye; and wherein the “dead” control comprises a portion of the sample that is fixed and washed and has been exposed to a viability dye.

[0082] Strain-Specific Controls: While the community-based control method works well in most cases, the unequal representation of taxa across a community can sometimes limit the availability of sufficient control cells for accurate viability measurements. For applications like drug product development, where pure cultures of individual strains are available, a separate13581226294Attorney Docket No.: 273089 / KBS-012WO / 577219approach can be employed. Each pure stock can be used to generate both live and dead controls in the presence of viability dye. This information is then used to build a reference database, against which experimental samples can be compared to ensure accurate viability assessments.

[0083] In some embodiments, the Strain-Specific Control comprises a preparation of “live” and “dead” pure culture of a known, individual strain, wherein the “live” pure culture comprises a portion of a culture of a known, individual strain that is fixed and washed and has not been exposed to a viability dye; and wherein the “dead” pure culture comprises a portion of a culture of a known, individual strain that is fixed and washed and has been exposed to a viability dye.

[0084] In some embodiments, the method further comprises (e.2) performing a second identifying assay, wherein the second identifying assay produces a fourth detectable signal.

[0085] In some embodiments, the fourth detectable signal derives from the second identifying assay and is indicative of a known gene, a known protein, or a known marker of cell stress or cell viability. In some embodiments, the fourth detectable signal derives from the second identifying assay and is indicative of a known gene. In some embodiments, the fourth detectable signal derives from the second identifying assay and is indicative of a known protein.

[0086] In some embodiments, the method further comprises (g) assessing the morphology of the cells.

[0087] In some embodiments, the morphology assessment comprises assessing the area, volume, specific radius, Euler number, solidity, eccentricity, perimeter, convex area, orientation relative to neighbors, geometry, or shape of the cell.

[0088] In some embodiments, the method further comprises (g.1.1) comparing the morphology assessments with reference data of known cells in a natural or healthy states to assess the health of the cells.

[0089] In some embodiments, the method further comprises (g.1.2) comparing the morphology assessments with reference data of known cells in an unnatural, stressed, diseased, or dead state to assess the health of the cells.Analysis

[0090] In some embodiments, the imaging with a microscope produces a first, second, and third detectable signal,14581226294Attorney Docket No.: 273089 / KBS-012WO / 577219wherein the first detectable signal derives from the viability dye and is indicative of the presence of dead cells;wherein the second detectable signal derives from the nuclear dye and is indicative of the presence of a viable cell fraction; andwherein the third detectable signal derives from the identifying assay and is indicative of a known microbial strain.

[0091] In some embodiments, the first detectable signal derives from the viability dye and is indicative of the presence of dead cells. In some embodiments, the viability dye is imaged by measuring emission spectra emitted from the viability dye. In some embodiments, the first detectable signal is a fluorescent signal at a specific wavelength (nm).

[0092] In some embodiments, the first detectable signal derives from the viability dye and is indicative of the presence of dead cells. In some embodiments, the viability dye is imaged by measuring emission spectra emitted from the viability dye. In some embodiments, the first detectable signal is a fluorescent signal at a specific wavelength (nm).

[0093] In some embodiments, the viability dye comprises LIVE / DEAD® Fixable Blue stain, LIVE / DEAD® Fixable Violet stain, LIVE / DEAD® Fixable Lime stain, LIVE / DEAD® Fixable Aqua stain, LIVE / DEAD® Fixable Yellow stain, LIVE / DEAD® Fixable Green stain, LIVE / DEAD® Fixable Olive stain, LIVE / DEAD® Fixable Orange stain, LIVE / DEAD® Fixable Red stain, LIVE / DEAD® Fixable Far Red stain, LIVE / DEAD® Fixable Scarlet stain, LIVE / DEAD® Fixable Near-IR (775) stain, LIVE / DEAD® Fixable Near-IR (780) stain, LIVE / DEAD® Fixable Near IR (876) stain, or 5-Cyano-2,3-ditolyl tetrazolium chloride (CTC).

[0094] In some embodiments, the viability dye is a LIVE / DEAD®™ Fixable Blue stain. In some embodiments, the viability dye is a LIVE / DEAD® Fixable Violet stain. In some embodiments, the viability dye is a LIVE / DEAD® Fixable Lime stain. In some embodiments, the viability dye is a LIVE / DEAD® Fixable Aqua stain. In some embodiments, the viability dye is a LIVE / DEAD® Fixable Yellow stain. In some embodiments, the viability dye is a LIVE / DEAD® Fixable Green stain. In some embodiments, the viability dye is a LIVE / DEAD® Fixable Olive stain. In some embodiments, the viability dye is a LIVE / DEAD® Fixable Orange stain. In some embodiments, the viability dye is a LIVE / DEAD® Fixable Red stain. In some embodiments, the viability dye is a LIVE / DEAD® Fixable Far Red stain. In some embodiments, the viability dye is a LIVE / DEAD® Fixable Scarlet stain. In some embodiments, the viability dye is a LIVE / DEAD®15581226294Attorney Docket No.: 273089 / KBS-012WO / 577219Fixable Near-IR (775) stain. In some embodiments, the viability dye is a LIVE / DEAD® Fixable Near-IR (780) stain. In some embodiments, the viability dye is a LIVE / DEAD® Fixable Near IR (876) stain. In some embodiments, the viability dye is 5-Cyano-2,3-ditolyl tetrazolium chloride (CTC). LIVE / DEAD® Fixable stains are commercially available from ThermoFisher Scientific.

[0095] In some embodiments, the second detectable signal derives from the nuclear dye and is indicative of the presence of a viable cell fraction. In some embodiments, the nuclear dye is imaged by measuring emission spectra emitted from the nuclear dye. In some embodiments, the second detectable signal is a fluorescent signal at a specific wavelength (nm).

[0096] In some embodiments, the nuclear dye comprises the nuclear dye comprises 4',6-diamidino-2-phenylindole (DAPI).

[0097] In some embodiments, the third detectable signal derives from the identifying assay and is indicative of a known microbial strain.

[0098] In some embodiments, the identifying assay comprises:(A) providing a first set of probes,wherein the first set of probes comprises at least one first encoding probe and at least one first emissive readout probe; and(B) contacting the first set of probes with the fixed cells to form a first complex; wherein the at least one first emissive readout probe emits a first color, and wherein the first color is assigned to a known microbial strain.

[0099] In some embodiments, the second identifying assay is indicative of a known gene, and wherein the second identifying assay comprises:contacting at least one second encoding probe with the cell to produce a second complex, wherein each encoding probe comprises an mRNA targeting sequence and an initiator sequence;adding two different DNA amplifier sequences to the second complex to produce a third complex, wherein each DNA amplifier comprises an initiator complimentary sequence and a readout sequence; andadding two second emissive readout probes to the third complex, wherein each second emissive readout probe comprises a fluorophore and a complimentary sequence to the second readout sequence of a corresponding DNA amplifier sequence.

[0100] In some embodiments, the second identifying assay is indicative of a known gene, and comprises performing a HiPR-Cycle assay as developed by the present applicant and as described16581226294Attorney Docket No.: 273089 / KBS-012WO / 577219in PCT Publication No. WO 2023 / 159240. The contents of which are incorporated by reference in their entirety.

[0101] In some embodiments, the second identifying assay is indicative of a known gene (e.g., HiPR-Cycle), and wherein the second identifying assay can include contacting at least one second encoding probe with sample or a cell to produce a second complex, adding at least two different DNA amplifiers to the second complex to produce a third complex, and adding second emissive readout probes to the third complex. Each second encoding probe can include a targeting sequence and an initiator sequence. Each DNA amplifier can include an initiator complimentary sequence and a readout sequence. Each second emissive readout probe can include a label and a complimentary sequence to the readout sequence of a corresponding DNA amplifier.

[0102] In some embodiments, the second identifying assay is indicative of a known gene (e.g., HiPR-Cycle), and wherein the second identifying assay can include generating a set of second probes, wherein each second probe includes:(i) a targeting sequence;(ii) at least one initiator sequence; and(iii) at least two DNA amplifiers, wherein each DNA amplifier includes an initiator complimentary sequence and a readout sequence;contacting the second set of probes with a sample or a cell to permit hybridization of the probes to nucleotides present in the sample or cell to produce a complex;adding a set of second emissive readout probes to the complex, wherein each second emissive readout probe includes a label and a sequence complimentary to the readout sequence of a corresponding DNA amplifier;detecting the emissive readout probes in the sample or cell;determining the spectra of “signal” (such as, e.g., puncta, blobs) and assigning them to a bacterium; anddecoding the spectra into a single, targeted transcript through means of signal deconvolution, error correction, comparison to reference standards.

[0103] In some embodiments, the second identifying assay is indicative of a known protein, and wherein the second identifying assay comprises: identifying the presence of immunoglobulins on the surface of the cell, wherein the identifying the presence of immunoglobulins on the surface of a cell comprises incorporating an oligonucleotide-conjugated antibody or a fluorophore17581226294Attorney Docket No.: 273089 / KBS-012WO / 577219conjugated antibody.Encoding Probe Hybridization

[0104] The methods described herein include providing a first set of probes. In some embodiments, the set of probes comprises at least one encoding probe and at least one emissive readout probe. In some embodiments, the first set of probes comprises at least one first encoding probe and at least one first emissive readout probe.

[0105] In some embodiments, the second encoding probe comprises an mRNA targeting sequence and an initiator sequence. The second encoding probes are probes that bind directly to a target or targeting sequence and contain either 1 or 2 branches extending away from the hybridization site. The branches can either correspond to the readout sequences, initiator sequences, and / or sequences that comprise at least one site for secondary hybridization events.

[0106] Encoding probes, for example, are designed to target bacterial ribosomal RNA (rRNA) and messenger RNA (mRNA) targets or a synthetic sequence.

[0107] For example, rRNA-probes can contain (5’ to 3’):a. Primer sequences to enrich probe pool.b. A readout-complementary sequence.c. rRNA target complementary sequence.d. A readout-complementary sequence (can be same or different than b). e. Primer sequences to enrich probe pool.

[0108] mRNA-probes contain (5’ to 3’):a. Primer sequences to enrich probe pool.b. An initiator sequence.c. mRNA target complementary sequence.d. An initiator sequence (can be same or different than b).e. Primer sequences to enrich probe pool.

[0109] Primer Sequences

[0110] In some embodiments, the primer sequence can include about 10 to about 30, about 15 to about 25, about 18 to about 23, about 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides long.

[0111] Targeting Sequence

[0112] In some embodiments, each of the encoding probes comprise a targeting sequence.

[0113] In some embodiments, the targeting sequence targets at least one of messenger RNA (mRNA), micro RNA (miRNA), long non coding RNA (IncRNA), ribosomal RNA (rRNA), small18581226294Attorney Docket No.: 273089 / KBS-012WO / 577219interfering RNA (siRNA), transfer RNA (tRNA), Crispr RNA (crRNA), trans-activating crispr RNA (tracrRNA), mitochondria RNA, Intronic RNA, viral mRNA, viral genomic RNA, environmental RNA, double-stranded RNA (dsRNA), small nuclear RNA (snRNA), small nucleolar (snoRNA), piwi-interacting RNA (piRNA), genomic DNA, synthetic DNA, DNA, plasmid DNA, a plasmid, viral DNA, retroviral DNA, environmental DNA, extracellular DNA, a protein, a small molecule, or an antigenic target. In some embodiments, the target is mRNA. In some embodiments, the target is rRNA. In some embodiments, the target is mRNA and rRNA.

[0114] In some embodiments, the targeting sequence targets messenger RNA (mRNA). In some embodiments, the targeting sequence targets micro RNA (miRNA). In some embodiments, the targeting sequence targets long non-coding RNA (IncRNA). In some embodiments, the targeting sequence targets ribosomal RNA (rRNA). In some embodiments, the targeting sequence targets small interfering RNA (siRNA). In some embodiments, the targeting sequence targets transfer RNA (tRNA). In some embodiments, the targeting sequence targets Crispr RNA (crRNA). In some embodiments, the targeting sequence targets trans-activating Crispr RNA (tracrRNA). In some embodiments, the targeting sequence targets mitochondrial RNA. In some embodiments, the targeting sequence targets intronic RNA. In some embodiments, the targeting sequence targets viral mRNA. In some embodiments, the targeting sequence targets viral genomic RNA. In some embodiments, the targeting sequence targets environmental RNA. In some embodiments, the targeting sequence targets double-stranded RNA (dsRNA). In some embodiments, the targeting sequence targets small nuclear RNA (snRNA). In some embodiments, the targeting sequence targets small nucleolar RNA (snoRNA). In some embodiments, the targeting sequence targets piwi-interacting RNA (piRNA). In some embodiments, the targeting sequence targets genomic DNA. In some embodiments, the targeting sequence targets synthetic DNA. In some embodiments, the targeting sequence targets DNA. In some embodiments, the targeting sequence targets plasmid DNA. In some embodiments, the targeting sequence targets a plasmid. In some embodiments, the targeting sequence targets viral DNA. In some embodiments, the targeting sequence targets retroviral DNA. In some embodiments, the targeting sequence targets environmental DNA. In some embodiments, the targeting sequence targets extracellular DNA. In some embodiments, the targeting sequence targets a protein. In some embodiments, the targeting sequence targets a small molecule. In some embodiments, the targeting sequence targets an antigenic target.

[0115] In some embodiments, the targeting sequence targets mRNA and / or rRNA. In some19581226294Attorney Docket No.: 273089 / KBS-012WO / 577219embodiments, the targeting sequence targets mRNA. In some embodiments, the targeting sequence targets rRNA. In some embodiments, the targeting sequence targets a 16S rRNA sequence, a 5S rRNA sequence, and / or a 23 S rRNA sequence in the microbial strain. In some embodiments, the targeting sequence targets a 16S rRNA sequence, a 5S rRNA sequence, and / or a 23S rRNA sequence in a bacterial strain. In some embodiments, the targeting sequence targets a 16S rRNA sequence, a 5S rRNA sequence, and / or a 23S rRNA sequence in the pathogen.

[0116] In some embodiments, the targeting sequence targets a 18S rRNA sequence, a 5.8S rRNA sequence, and / or a 28S rRNA sequence in the in the microbial strain, wherein the microbial strain is a eukaryotic microbial strain. In some embodiments, the targeting sequence targets a 18S rRNA sequence. In some embodiments, the targeting sequence targets a 5.8S rRNA sequence. In some embodiments, the targeting sequence targets a 28S rRNA sequence.

[0117] In some embodiments, the targeting sequence of the encoding probe is substantially complementary to a specific target sequence. By “substantially complementary” it is meant that the nucleic acid fragment is capable of hybridizing to at least one nucleic acid strand or duplex even if less than all nucleobases do not base pair with a counterpart nucleobase.

[0118] In some embodiments, the targeting sequence is designed to have a predicted melting temperature of between about 55 °C and about 65 °C. In some embodiments, the predicted melting temperature of the targeting sequence is 55 °C, 56 °C, 57 °C, 58 °C, 59 °C, 60 °C, 61 °C, 62 °C, 63 °C, 64 °C or 65 °C. In some embodiments, the targeting sequence can have a GC content of about 55%, 60%, 65% or 70%.

[0119] In some embodiments, the targeting sequence can include about 10 to about 35, about 15 to about 30, about 18 to about 30, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long.

[0120] In some embodiments, to hybridize the encoding probes to the complex, encoding buffer is added to the sample. In some embodiments, the encoding buffer can include a denaturing / deionizing agent, a salt buffer, a detergent, a polyanionic polymer, a blocking agent, or combinations thereof. In some embodiments, the encoding buffer includes more than one type of agent, for example, the encoding buffer can include two or more polyanionic polymers and / or two or more blocking agents.

[0121] Initiator Sequence

[0122] In some embodiments, the second encoding probe can include the initiator sequence on20581226294Attorney Docket No.: 273089 / KBS-012WO / 577219the 5’ end and / or the 3’ end. In some embodiments, the second encoding probe can include an initiator sequence on the 5’ end. In some embodiments, the second encoding probe can include an initiator sequence on the 3’ end. In some embodiments, the second encoding probe can include an initiator sequence on the 5’ end and an initiator sequence on the 3’ end. In some embodiments, the two initiator sequences have different sequences. In some embodiments, the two initiator sequences have the same sequence. In some embodiments, the second encoding probe can include the at least one sequence that comprise at least one site for secondary hybridization events on the 5’ end and / or the 3’ end.

[0123] In some embodiments, the initiator sequence is about 10 to about 30, about 15 to about 25, about 18 to about 23, about 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides long. In some embodiments, the initiator sequence is substantially complementary to the toehold sequence of the DNA amplifier.

[0124] In some embodiments, a second encoding probe can include two fractional second encoding probes that have neighboring target regions. The two fractional second encoding probes bind to the target to colocalize a full initiator. The colocalized full initiator is required to initiate the hybridization chain reaction by a corresponding amplifier. In some embodiments, there is an energetically unfavorable junction between the two duplexes. In some embodiments, by configuring the fractional initiators to bind to overlapping regions of the amplifier, the duplex can relax into an energetically more favorable conformation, increasing the affinity between the colocalized full initiator and the amplifier. In some embodiments the affinity between the two encoding probes and the target can be increased by configuring the target-binding regions of the two encoding probes to bind to overlapping regions of the target so as to permit the junction between the molecules to relax to an energetically favorable conformation. In some embodiments, the two fractional second encoding probes have about the same nucleotide length. In some embodiments, the two fractional second encoding probes have different nucleotide lengths, for example one fractional second encoding probe may have about 25% nucleotide length and the other the 75% nucleotide length.

[0125] DNA Amplifier Sequences

[0126] ‘ ‘DNA amplifiers,” “amplifiers,” and “amplifier sequences” are used interchangeably when referring to the HiPR-Cycle method described herein.

[0127] Amplifier sequences are metastable hairpin sequences that come in pairs (###_H1 and21581226294Attorney Docket No.: 273089 / KBS-012WO / 577219###_H2), the design is based on HCR amplifier probes and contains a readout-complementary sequencing at the 5 ’-end (in the case of ###_H1) or 3 ’-end (###_H2), adjacent to the initiator sequence. Amplifier sequences are stored in a high salt buffer (e.g., 120 mM NaCl), and are heated (e.g., 95 °C for 1.5 min) and annealed (e.g., room temperature for 30 min) prior to addition to the sample.

[0128] In some embodiments the amplifiers are stored in high salt buffer, such as, 100 mM, 120 mM, 200 mM, 250 mM, 500 mM, 750 mM, or 1 M NaCl. In some embodiments, the amplifier sequences are heated at high temperatures (e.g., about 95 °C to 100 °C) for a short period of time (e.g., 1, 2, 3, 4 or 5 minutes) followed by a cooling period of about 15 min to 1 hour, e.g., 30 min, to room temperature.

[0129] In some embodiments, at least two amplifier probes (one pair) are used for at least one readout probe. In some embodiments, at least two amplifier probes (one pair) are used for multiple (e.g., two or more) readout probes. In some embodiments, at least two amplifier probes (one pair) are used for each readout probe. For example, each amplifier probe can have:a. A readout complementary sequence (15-20 nt).b. An optional first spacer sequence (0-5 nt).c. A toehold sequence. (9nt)d. A stem sequence.e. A loop sequence (9 nt) complementary to the initiator on the paired amplifier). f. A stem-complementary sequence.

[0130] In other examples, each amplifier probe can have:a. A readout complementary sequence (15-20 nt).b. An optional first spacer sequence (0-5 nt).c. A toehold sequence. (9nt)d. A stem sequence.e. An optional second spacer sequence (0-5 nt).f. A loop sequence (9 nt) complementary to the initiator on the paired amplifier). g. A stem-complementary sequence.

[0131] In other examples, each amplifier probe can have:a. A stem-complementary sequence.b. A loop sequence (9 nt) complementary to the initiator on the paired amplifier).22581226294Attorney Docket No.: 273089 / KBS-012WO / 577219c. An optional second spacer sequence (0-5 nt).d. A stem sequence.e. A toehold sequence (9nt)f. An optional first spacer sequence (0-5 nt).g. A readout complementary sequence (15-20 nt).

[0132] The readout complementary sequence of the amplifier probe / DNA amplifier is a nucleotide sequence that is about 10 to about 25, about 15 to about 20, about 15, 16, 17, 18, 19, or 20 nucleotides long and has a nucleotide sequence that is the complement of the emissive readout probe sequence. In some embodiments, the readout sequence present in the amplifier probe is also known as a “landing pad sequence.” In some embodiments, the readout complementary sequence present in the amplifier probe is also known as a “landing pad sequence.”

[0133] Each of the optional first and second spacer sequences of the amplifier probe / DNA amplifier is about 1 to 5, about 1, 2, 3, 4, or 5 nucleotides long.

[0134] The toehold sequence of the amplifier probe / DNA amplifier is a nucleotide sequence that is about 10 to about 30, about 15 to about 25, about 18 to about 23, about 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides long and has a nucleotide sequence that is the complement to the initiator sequence of the encoding probe.

[0135] The stem sequence of the amplifier probe / DNA amplifier is a nucleotide sequence that is about 5 to about 15, about 7 to about 10, about 5, 6, 7, 8, 9, or 10 nucleotides long and has a nucleotide sequence that is a complement to its other stem.

[0136] The loop sequence of the amplifier probe / DNA amplifier is a nucleotide sequence that is about 5 to about 15, about 7 to about 10, about 5, 6, 7, 8, 9, or 10 nucleotides long and has a nucleotide sequence that is a complement to the toehold sequence of its pair DNA amplifier.

[0137] The stem-complimentary sequence of the amplifier probe / DNA amplifier is a nucleotide sequence that is about 5 to about 15, about 7 to about 10, about 5, 6, 7, 8, 9, or 10 nucleotides long and has a nucleotide sequence that is a complement to its other stem.

[0138] In some embodiments, one of the two DNA amplifiers can include, from 5’ to 3’, a readout sequence (R.1), a toehold sequence (T.l), a stem sequence (S.l), a loop sequence (L.l), and a complement stem sequence (cS.l). In some embodiments, one of the two DNA amplifiers can include, from 5’ to 3’, a readout sequence (R.l), a first spacer sequence (Sp.1-1), a toehold sequence (T.l), a stem sequence (S.l), a loop sequence (L.l), and a complement stem sequence23581226294Attorney Docket No.: 273089 / KBS-012WO / 577219(cS.1). In some embodiments, one of the two DNA amplifiers can include, from 5’ to 3’, a readout sequence (R.l), a first spacer sequence (Sp.1-1), a toehold sequence (T.l), a stem sequence (S.l), a second spacer sequence (Sp.1-2), a loop sequence (L.l), and a complement stem sequence (cS.l).

[0139] In some embodiments, one of the two DNA amplifiers can include, from 5’ to 3’, a stem sequence (S.2), a loop sequence (L.2), a complement stem sequence (cS.2), a toehold sequence (T.2), and a readout sequence (R.2).

[0140] In some embodiments, one of the two DNA amplifiers can include, from 5’ to 3’, a stem sequence (S.2), a loop sequence (L.2), a complement stem sequence (cS.2), a toehold sequence (T.2), a first spacer sequence (Sp.2-1), and a readout sequence (R.2). In some embodiments, one of the two DNA amplifiers can include, from 5’ to 3’, a stem sequence (S.2), a loop sequence (L.2), a second spacer sequence (Sp. 2-2), a complement stem sequence (cS.2), a toehold sequence (T.2), a first spacer sequence (Sp.2-1), and a readout sequence (R.2).

[0141] In some embodiments, one of the two DNA amplifiers can include, from 5’ to 3’, a toehold sequence (T.l), a stem sequence (S.l), a loop sequence (L.l), a complement stem sequence (cS.l), and a readout sequence (R.l). In some embodiments, one of the two DNA amplifiers can include, from 5’ to 3’, a readout sequence (R.2), a stem sequence (S.2), a loop sequence (L.2), a complement stem sequence (cS.2), and a toehold sequence (T.2).

[0142] In some embodiments, the DNA amplifiers can further include a first spacer sequence and / or a second spacer sequence. In some embodiments, the DNA amplifiers further can include a first spacer sequence. In some embodiments, the DNA amplifiers further can include a second spacer sequence. In some embodiments, the DNA amplifiers further can include a first spacer sequence and a second spacer sequence. In some embodiments, the first spacer sequence is on the 3’ end of the readout sequence and to the 5’ end of the toehold sequence of the DNA amplifier. In some embodiments, the first spacer sequence is on the 3’ end of the toehold sequence and to the 5’ end of the readout sequence of the DNA amplifier. In some embodiments, the first spacer sequence is 1, 2, 3, 4, or 5 nucleotides long. In some embodiments, the first spacer sequence is a random string of three nucleotides. In some embodiments, the second spacer sequence is on the 3’ end of the stem sequence and to the 5’ end of the loop sequence complementary to the initiator of the DNA amplifier. In some embodiments, the second spacer sequence is on the 3’ end of the loop sequence complementary to the initiator and to the 5’ end of the stem sequence of the DNA amplifier. In some embodiments, the second spacer sequence is 1, 2, 3, 4, or 5 nucleotides long.24581226294Attorney Docket No.: 273089 / KBS-012WO / 577219In some embodiments, the second spacer sequence is a random string of three nucleotides.

[0143] In some embodiments, the readout sequence of the DNA amplifier can include 15 to 30 nucleotides, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the readout sequence of each DNA amplifier is the same sequence. In some embodiments, the readout sequence of each DNA amplifier is the different. In some embodiments, the readout sequence of DNA amplifier has a 50% or less sequence identity to the other the readout sequence of DNA amplifier.

[0144] In some embodiments, the toehold sequence (T.l) is a sequence complementary to the loop sequence (L.2) of the other DNA amplifier. In some embodiments, the loop sequence (L.1 ) is a sequence complementary to the toehold sequence (T.2) of the other DNA amplifier. In some embodiments, the toehold sequence is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides long. In some embodiments, the loop sequence is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides long. In some embodiments, the stem region and its complementary sequence are each 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides long.

[0145] In some embodiments, the second identifying assay can include adding four DNA amplifiers.

[0146] In some embodiments, one of the four DNA amplifiers can include, from 5’ to 3’ a amplifier initiator sequence (HI.l), a toehold sequence (T.l), a stem sequence (S.l), a loop sequence (L.l), and a complement stem sequence (cS.l). In some embodiments, one of the four DNA amplifiers can include, from 5’ to 3’ a stem sequence (S.2), a loop sequence (L.2), complement stem sequence (cS.2), a toehold sequence (T.2), and an amplifier initiator sequence (HI.2). In some embodiments, one of the four DNA amplifiers can include, from 5’ to 3’, a readout sequence (R.l-2), a toehold sequence (T.l-2), a stem sequence (S.1-2), a loop sequence (L.l-2), and a complement stem sequence (cS.1-2). In some embodiments, one of the four DNA amplifiers can include, from 5’ to 3’, a stem sequence (S.2-1), a loop sequence (L.2-1), a complement stem sequence (cS.2-1), a toehold sequence (T.2-1), and a readout sequence (R.2-1).

[0147] In some embodiments, the four DNA amplifiers can further include a first and / or second spacer sequence, wherein the first and / or second spacer sequence is about 1 to 5, about 1, 2, 3, 4, or 5 nucleotides long.

[0148] In some embodiments, the amplifier initiator sequence (HI.l) is a sequence complementary to the loop sequence (L.l-2 or L.2-1) of one of the other DNA amplifiers can25581226294Attorney Docket No.: 273089 / KBS-012WO / 577219include the readout sequence. In some embodiments, the toehold sequence (T.l) is a sequence complementary to the loop sequence (L.2) of the other DNA amplifier can include the amplifier initiator sequence. In some embodiments, the loop sequence (L.1) is a sequence complementary to the toehold sequence (T.2) of the other DNA amplifier can include the amplifier initiator sequence. In some embodiments, the amplifier initiator sequence is unique so that its sequence is not complementary to any other sequence. In this instance, the initiator sequence is different from the rest of the sequences so that it does not prematurely trigger the amplification reaction.

[0149] Readout Probe Hybridization

[0150] After the encoding hybridization step is complete, the methods described herein acquire one or more emission spectra from the at least one first emissive readout probe.

[0151] In some embodiments of the second identifying assay (e.g., HiPR-Cycle), readout probes bind to the DNA amplifier complexes that form in the assay. They can be added during or after the amplification step.

[0152] In some embodiments, each emissive readout probe can include a label and a complementary sequence to the readout sequence of the first encoding probe. In some embodiments, this step may be referred to as the “readout probe hybridization” step. In here, the emissive readout probes hybridize to their complementary sequences present in the complexes formed.

[0153] Emissive readouts probes are 10-50 nucleotide-long oligonucleotides bound with one of eleven fluorescent dyes at the 5’ - and / or 3’ - end.

[0154] Readout probes can be designed as follows:a. Are coupled to 1, 2, or more fluorescent dyes.b. Are orthogonal to all biological sequences.c. Are orthogonal to each other / each other’s complementary sequences.

[0155] In some embodiments, the emissive readout sequence is about 10 to about 50, about 15 to about 50, about 15 to about 45, about 15 to about 35, about 15 to about 30, about 18 to about 24, about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides long.

[0156] In some embodiments, the emissive readout probe can include a label on the 5’ or 3’ end. In some embodiments, the emissive readout probe can include a label on the 5’ end and a label on the 3’ end. In some embodiments, the labels are the same. In some embodiments, the26581226294Attorney Docket No.: 273089 / KBS-012WO / 577219labels are different.

[0157] In some embodiments, the label is a fluorescent entity (fluorophore) or phosphorescent entity. In some embodiments, the label is a cyanine dye (e.g., Cy2, Cy3, Cy3B, Cy5, Cy5.5, Cy7, etc.), Alexa Fluor dye, Atto dye, photoswitchable dye, photoactivatable dye, fluorescent dye, metal nanoparticle, semiconductor nanoparticle or “quantum dots”, fluorescent protein such as GFP (Green Fluorescent Protein), or photoactivatable fluorescent protein, such as PAGFP, PSCFP, PSCFP2, Dendra, Dendra2, EosFP, tdEos, mEos2, mEos3, PAmCherry, PAtagRFP, mMaple, mMaple2, and mMaple3.

[0158] In some embodiments, the label is Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 561, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 647-R-phycoerythrin, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 680-allophycocyanin, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, Alexa Fluor Plus 405, Alexa Fluor Plus 488, Alexa Fluor Plus 555, Alexa Fluor Plus 594, Alexa Fluor Plus 647, Alexa Fluor Plus 680, Alexa Fluor Plus 750, Alexa Fluor Plus 800, Pacific Blue, Pacific Green, Rhodamine Red X, DyLight 485-LS, DyLight-510-LS, DyLight 515-LS, DyLight 521-LS, Hydroxycoumarin, methoxycoumarin, Cy2, FAM, Fluorescein FITC, R-phycoerythrin (PE), Tamara, Cy3.5581 , Rox, Red 613, Texas Red, Cy5, Cy5.5, Cy7, Allophycocyanin, ATTO 430LS, ATTO 490LS, ATTO 390, ATTO 425, Cyan 500 NHS-Ester, ATTO 465, ATTO 488, ATTO 495, ATTO RhollO, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rholl, ATTO Rhol2, ATTO Thiol2, ATTO RholOl, ATTO 590, ATTO 594, ATTO Rhol3, ATTO 610, ATTO 620, ATTO Rhol4, ATTO 633, ATTO 643, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxal2, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740.

[0159] In some embodiments, the label is Alexa Fluor 350. In some embodiments, the label is Alexa Fluor 405. In some embodiments, the label is Alexa Fluor 430. In some embodiments, the label is Alexa Fluor 488. In some embodiments, the label is Alexa Fluor 514. In some embodiments, the label is Alexa Fluor 532. In some embodiments, the label is Alexa Fluor 546. In some embodiments, the label is Alexa Fluor 555. In some embodiments, the label is Alexa Fluor 561. In some embodiments, the label is Alexa Fluor 568. In some embodiments, the label is Alexa Fluor 594. In some embodiments, the label is Alexa Fluor 610. In some embodiments, the label is27581226294Attorney Docket No.: 273089 / KBS-012WO / 577219Alexa Fluor 633. In some embodiments, the label is Alexa Fluor 635. In some embodiments, the label is Alexa Fluor 647. In some embodiments, the label is Alexa Fluor 647-R-phycoerythrin. In some embodiments, the label is Alexa Fluor 660. In some embodiments, the label is Alexa Fluor 680. In some embodiments, the label is Alexa Fluor 680-allophycocyanin. In some embodiments, the label is Alexa Fluor 700. In some embodiments, the label is Alexa Fluor 750. In some embodiments, the label is Alexa Fluor 790. In some embodiments, the label is Alexa Fluor Plus 405. In some embodiments, the label is Alexa Fluor Plus 488. In some embodiments, the label is Alexa Fluor Plus 555. In some embodiments, the label is Alexa Fluor Plus 594. In some embodiments, the label is Alexa Fluor Plus 647. In some embodiments, the label is Alexa Fluor Plus 680. In some embodiments, the label is Alexa Fluor Plus 750. In some embodiments, the label is Alexa Fluor Plus 800. In some embodiments, the label is Pacific Blue. In some embodiments, the label is Pacific Green. In some embodiments, the label is Rhodamine Red X. In some embodiments, the label is DyLight 485-LS. In some embodiments, the label is DyLight-510-LS. In some embodiments, the label is DyLight 515-LS. In some embodiments, the label is DyLight 521-LS. In some embodiments, the label is Hydroxycoumarin. In some embodiments, the label is methoxycoumarin. In some embodiments, the label is Cy2. In some embodiments, the label is FAM. In some embodiments, the label is Fluorescein FITC. In some embodiments, the label is R-phycoerythrin (PE). In some embodiments, the label is Tamara. In some embodiments, the label is Cy3.5 581. In some embodiments, the label is Rox. In some embodiments, the label is Red 613. In some embodiments, the label is Texas Red. In some embodiments, the label is Cy5. In some embodiments, the label is Cy5.5. In some embodiments, the label is Cy7. In some embodiments, the label is Allophycocyanin. In some embodiments, the label is ATTO 430LS. In some embodiments, the label is ATTO 490LS. In some embodiments, the label is ATTO 390. In some embodiments, the label is ATTO 425. In some embodiments, the label is Cyan 500 NHS-Ester. In some embodiments, the label is ATTO 465. In some embodiments, the label is ATTO 488. In some embodiments, the label is ATTO 495. In some embodiments, the label is ATTO Rhol 10. In some embodiments, the label is ATTO 514. In some embodiments, the label is ATTO 520. In some embodiments, the label is ATTO 532. In some embodiments, the label is ATTO Rho6G. In some embodiments, the label is ATTO 542. In some embodiments, the label is ATTO 550. In some embodiments, the label is ATTO 565. In some embodiments, the label is ATTO Rho3B. In some embodiments, the label is ATTO Rhol 1. In some embodiments, the label is ATTO Rhol2. In some28581226294Attorney Docket No.: 273089 / KBS-012WO / 577219embodiments, the label is ATTO Thiol2. In some embodiments, the label is ATTO RholOl. In some embodiments, the label is ATTO 590. In some embodiments, the label is ATTO 594. In some embodiments, the label is ATTO Rhol3. In some embodiments, the label is ATTO 610. In some embodiments, the label is ATTO 620. In some embodiments, the label is ATTO Rhol4. In some embodiments, the label is ATTO 633. In some embodiments, the label is ATTO 643. In some embodiments, the label is ATTO 647. In some embodiments, the label is ATTO 647N. In some embodiments, the label is ATTO 655. In some embodiments, the label is ATTO Oxal2. In some embodiments, the label is ATTO 665. In some embodiments, the label is ATTO 680. In some embodiments, the label is ATTO 700. In some embodiments, the label is ATTO 725. In some embodiments, the label is ATTO 740.

[0160] In some embodiments, to hybridize the readout probes, readout buffer is added to the sample. In some embodiments, the readout buffer can include a denaturing / deionizing agent, a salt buffer, a detergent, a polyanionic polymer, a blocking agent, or combinations thereof. In some embodiments, the readout buffer includes more than one type of agent, for example, the readout buffer can include two or more polyanionic polymers and / or two or more blocking agents.

[0161] In some embodiments, after each reaction and before proceeding to the next one, the samples or probes are washed with a “wash buffer.”

[0162] In some embodiments, the wash buffer can include a denaturing / deionizing agent, a salt buffer, a detergent, a polyanionic polymer, acids, a pH stabilizer, a chelating agent, or combinations thereof. In some embodiments, the wash buffer can include more than one type of agent, for example, the wash buffer can include two or more detergents. In some embodiments, the wash buffer can include a denaturing / deionizing agent, a salt buffer, a detergent, a polyanionic polymer, and an acid. In some embodiments, the wash buffer can include a salt buffer and a detergent. In some embodiments, the wash buffer can include a salt buffer, a pH stabilizer, and a chelating agent.

[0163] In some embodiments, the encoding probes are imaged by measuring emission spectra emitted from the readout probes. In some embodiments, the methods comprise acquiring one or more emission spectra with a confocal microscope from the at least one emissive readout probe. In some embodiments, the methods determine the spectra of signal and assigning them to a species of the microbial strain. In some embodiments, the methods comprise decoding the spectra into a single barcode representative of a specific taxa or microbial strain through means of signal29581226294Attorney Docket No.: 273089 / KBS-012WO / 577219deconvolution, error correction, comparison to reference standards.

[0164] In some embodiments, the emissive readout probes are imaged using hyperspectral microscopy, widefield epifluorescence microscopy, widefield microscopy, or whole-slide scanner.

[0165] In some embodiments, the emissive readout probe is imaged using point scanning confocal microscopy, spinning disk confocal microscopy, lattice lightsheet microscopy, or light field microscopy.

[0166] In some embodiments, the microscope is a hyperspectral microscope, widefield epifluorescence microscope, widefield microscope, whole-slide scanner, point scanning confocal microscope, spinning disk confocal microscope, lattice lightsheet microscope, or light field microscope.

[0167] In some embodiments, the imaging with the above modalities can be imaged at different spatial resolution using different microscope objectives including lOx, 20x, 32x, 40x, 63x, or lOOx, which can be compatible with air, oil, or water immersion medium.

[0168] In some embodiments, the detection strategy used is channel, spectral, channel and fluorescence lifetime, or spectral and fluorescence lifetime.

[0169] In some embodiments, the array is a microscope slide, at least one chamber, at least one microfluidic device, at least one well, at least one plate, at least one filter membrane, at least one cover slip, or at least one adhesive tape. In some embodiments, the array is a microscope slide. In some embodiments, the array is at least one chamber. In some embodiments, the array is at least one microfluidic device. In some embodiments, the array is at least one well. In some embodiments, the array is at least one plate. In some embodiments, the array is at least one filter membrane. In some embodiments, the array is at least one adhesive tape. In some embodiments, the array is at least one piece of adhesive tape.

[0170] In some embodiments, the array is pre-treated with a coating to enhance adhesion of microbes (i.e. poly-d-lysine).Barcoded Probes

[0171] Encoding Barcoded Probes

[0172] The encoding probes used in the methods described herein use encoding barcoded probes. The encoding barcoded probes represent a probe / sequence that is specific to a target sequence in the sample / complex with a unique code.30581226294Attorney Docket No.: 273089 / KBS-012WO / 577219

[0173] In some embodiments, the encoding barcoded probes include the encoding probes and readout sequences described herein.

[0174] In some embodiments, each sample or target in the sample to be identified is assigned a unique n-bit binary code selected from a plurality of unique n-bit binary codes, where n is an integer equal to or greater than 1. In some embodiments, each encoding probe does not have a unique binary code, instead groups of or all of the encoding probes have the same color.

[0175] A “binary code” refers to a representation of target sequence in a sample using a string made up of a plurality of “0” and “1” from the binary number system. The binary code is made up of a pattern of n binary digits (n-bits), where n is an integer representing the number of labels used. The bigger the number n, the greater number of targets can be represented using the binary code. For example, a binary code of eight bits (an 8-bit binary code, using 8 different labels) can represent up to 255 (28- 1) possible targets. (One is subtracted from the total possible number of codes because no target sequence is assigned a code of all zeros “00000000.” A code of all zeros would mean no decoding sequence, and thus no label, is attached. In other words, there are no nonlabeled target sequences.) Similarly, a binary code of ten bits (a 10-bit binary code) can represent up to 1023 (210- 1) possible target sequences. In some embodiments a binary code may be translated into and represented by a decimal number. For example, the 10-bit binary code “0001100001” can also be represented as the decimal number “97.”

[0176] Each digit in a unique encoding binary code represents whether a readout probe and the fluorophore corresponding to that readout probe are present for the selected strain or taxa. In some embodiments, each digit in the encoding binary code corresponds to a Readout probe (from Readout probe 1 (Rl) through Readout probe n (Rn) in an n-bit coding scheme). In a specific embodiment, the n is 10 and the digits of an n-bit code correspond to Rl through R10. In some embodiments, the fluorophores that correspond to Rl through Rn are determined arbitrarily. For example, n is 10, and Rl corresponds to an Alexa 488 fluorophore, R2 corresponds to an Alexa 546 fluorophore, R3 corresponds to a 6-ROX (6-Carboxy-X-Rhodamine, or Rhodamine Red X) fluorophore, R4 corresponds to a Alexa Fluor 594, R5 corresponds to a Alexa Fluor 660, R6 corresponds to an Alexa 610 fluorophore, R7 corresponds to an Alexa 647 fluorophore, R8 corresponds to a DyLight-510-LS fluorophore, R9 corresponds to an Alexa 405 fluorophore, and R10 corresponds to an Alex532 fluorophore. In some embodiments, other labels / fluorophores are used in the n-bit encoding system.31581226294Attorney Docket No.: 273089 / KBS-012WO / 577219

[0177] In some embodiments, the n-bit encoding binary code is selected from the group consisting of 2-bit binary code, 3-bit binary code, 4-bit binary code, 5-bit binary code, 6-bit binary code, 7 -bit binary code, 8-bit binary code, 9-bit binary code, 10-bit binary code, 11 -bit binary code, 12-bit binary code, 13-bit binary code, 14-bit binary code, 15-bit binary code, 16-bit binary code, 17-bit binary code, 18-bit binary code, 19-bit binary code, 20- bit binary code, 21 -bit binary code, 22-bit binary code, 23 -bit binary code, 24-bit binary code, 25-bit binary code, 26-bit binary code, 27-bit binary code, 28 bit binary code, 29- bit binary code, and 30-bit binary code.

[0178] Viability Barcoded Classification

[0179] The viability dye can be treated as a unique bit in a barcode system.

[0180] In some embodiments, dead cells register this bit as "1" and live cells as "0." The first digit of the cell barcode is reserved for viability. For example if a in a barcode represented by X101, the "101" represents taxonomic identification, such as E. coli. In this system, "0101" represents a live A', coli, while "1101" represents a dead A. coli.Sample

[0181] In some embodiments, the sample comprises at least one cell, a cell suspension, a tissue biopsy, a tissue specimen, bone biopsies, organoids, three-dimensional hydrogel scaffolds, transwell systems, or plant biopsies.

[0182] In some embodiments, the sample is a cell. In some embodiments, the cell is a bacterial cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a unicellular organism including protozoa, chromista, algae, or fungi. In some embodiments, the eukaryotic cell is part of a multicellular organism from chromista, plantae, fungi, or animalia. In some embodiments, the sample is a tissue composed of cells. In some embodiments, the cell contains foreign DNA / RNA from viruses, plasmids, and bacteria. In some embodiments, the cell has been genetically modified or synthetically engineered. In some embodiments, the cell has been genetically modified.

[0183] In some embodiments, the sample can include a plurality of cells. In some embodiments, each cell in the plurality of cells can include a specific targeting sequence, which may or may not be the same from the other targeting sequences.

[0184] In some embodiments, the sample is a whole organism.

[0185] In some embodiments, the sample comprises a plurality of cells. In some embodiments,32581226294Attorney Docket No.: 273089 / KBS-012WO / 577219the sample comprises a plurality of cells that contain the pathogen.

[0186] In some embodiments, the sample is obtained from a patient diagnosed with, or suspected to be suffering from an infection, disease, or disorder. In some embodiments, the patient has been diagnosed with, or is suspected to be suffering from a bacterial, viral, fungal, or parasitic infection. In some embodiments, the infection includes, but is not limited to, Acute Flaccid Myelitis, Anaplasmosis, Anthrax, Babesiosis, Botulism, Brucellosis, Campylobacteriosis, Carbapenem-resistant Infection (CRE / CRPA), Chancroid, Chickenpox, Chikungunya Virus Infection (Chikungunya), Chlamydia, Ciguatera (Harmful Algae Blooms (HABs)), Clostridium Difficile Infection, Clostridium Perfringens (Epsilon Toxin), Coccidioidomycosis fungal infection (Valley fever), COVID-19 (Coronavirus Disease 2019), Creutzfeldt- Jacob Disease, transmissible spongiform encephalopathy (CJD), Cryptosporidiosis (Crypto), Cyclosporiasis, Dengue, 1,2, 3, 4 (Dengue Fever), Diphtheria, E. coll infection, Shiga toxin-producing (STEC), Eastern Equine Encephalitis (EEE), Ebola Hemorrhagic Fever (Ebola), Ehrlichiosis, Encephalitis, Arboviral or parainfectious, Enterovirus Infection , D68 (EV-D68), Enterovirus Infection, Non-Polio (NonPolio Enterovirus), Giardiasis (Giardia), Glanders, Gonococcal Infection (Gonorrhea), Granuloma inguinale, Haemophilus Influenza disease, Type B (Hib or H-flu), Hantavirus Pulmonary Syndrome (HPS), Hemolytic Uremic Syndrome (HUS), Hepatitis (A, B, C, D, and / or E), Herpes Herpes Zoster, zoster VZV (Shingles), Histoplasmosis infection (Histoplasmosis), Human Immunodeficiency Virus / AIDS (HIV / AIDS), Human Papillomavirus (HPV), Influenza (Flu), Lead Poisoning, Legionellosis (Legionnaires Disease), Leishmaniasis, Leprosy (Hansens Disease), Leptospirosis, Listeriosis (Listeria), Lyme Disease, Lymphogranuloma venereum infection (LGV), Malaria, Measles, Melioidosis, Meningitis, Viral (Meningitis, viral), Meningococcal Disease , Bacterial (Meningitis, bacterial), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Mononucleosis, Multisystem Inflammatory Syndrome in Children (MIS-C), Mumps, Norovirus, Paralytic Shellfish Poisoning (Paralytic Shellfish Poisoning, Ciguatera), Pediculosis (Lice, Head and Body Lice), Pelvic Inflammatory Disease (PID), Pertussis (Whooping Cough), Plague; Bubonic, Septicemic, Pneumonic (Plague), Pneumococcal Disease (Pneumonia), Poliomyelitis (Polio), Powassan, Psittacosis (Parrot Fever), Phthiriasis (Crabs; Pubic Lice Infestation), Pustular Rash diseases (Small pox, monkeypox, cowpox), Q-Fever, Rabies, Ricin Poisoning, Rickettsiosis (Rocky Mountain Spotted Fever), Rubella, Salmonellosis gastroenteritis (Salmonella), Scabies Infestation (Scabies), Scombroid, Septic Shock (Sepsis),33581226294Attorney Docket No.: 273089 / KBS-012WO / 577219Severe Acute Respiratory Syndrome (SARS), Shigellosis gastroenteritis (Shigella), Smallpox, Staphylococcal Infection , Methicillin-resistant (MRSA), Staphylococcal Food Poisoning, Enterotoxin - B Poisoning (Staph Food Poisoning), Staphylococcal Infection, Vancomycin Intermediate (VISA), Staphylococcal Infection, Vancomycin Resistant (VRSA), Streptococcal Disease , Group A (invasive) (Strep A (invasive)), Streptococcal Disease, Group B (Strep-B), Streptococcal Toxic-Shock Syndrome, STSS, Toxic Shock (STSS, TSS), Syphilis , primary, secondary, early latent, late latent, congenital, Tetanus, Toxoplasmosis, Trichomoniasis (Trichomonas infection), Trichinosis Infection (Trichinosis), Tuberculosis (Latent) (LTBI), Tuberculosis (TB), Tularemia (Rabbit fever), Typhus, Typhoid Fever, Group D, Vaginosis , bacterial (Yeast Infection), Vaping-Associated Lung Injury (e-Cigarette Associated Lung Injury), Varicella (Chickenpox), Vibrio cholerae (Cholera), Vibriosis (Vibrio), Viral Hemorrhagic Fever (Ebola, Lassa, Marburg), West Nile Virus, Yellow Fever, Yersenia (Yersinia), or Zika Virus Infection (Zika).

[0187] In some embodiments, when the sample is obtained from a patient, the patient has been diagnosed with, or is suspected to be suffering from an infection caused by a bacterium selected from the group consisting of: Acinetobacter, Actinomyces, Aerococcus, Bacteroides, Bartonella, Brucella, Bordetella, Burkholderia, Campylobacter, Chlamydia, Citrobacter, Clostridium, Corynebacterium, Edwardsiella, Elizabethkingia, Enterobacter, Enterococcus, Escherichia, Fusobacterium, Haemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Listeria, Morganella, Mycobacterium, Mycoplasma, Neisseria, Pantoea, Prevotella, Proteus, Providencia, Pseudomonas, Raoultella, Salmonella, Serratia, Shigella, Staphylococcus, Stenotrophomonas, Streptococcus, Ureaplasma, and Vibrio.

[0188] In some embodiments, when the sample is obtained from a patient, the patient has been diagnosed with, or is suspected to be suffering from an infection caused by a virus selected from the group consisting of: bacteriophage, RNA bacteriophage (e.g., MS2, AP205, PP7 and QP), Infectious Haematopoietic Necrosis Virus, Parvovirus, Herpes Simplex Virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Measles virus, Mumps virus, Rubella virus, HIV, Influenza virus, Rhinovirus, Rotavirus A, Rotavirus B, Rotavirus C, Respiratory Syncytial Virus (RSV), Varicella zoster, and Poliovirus, Norovirus, Zika virus, Dengue Virus, Rabies Virus, Newcastle Disease Virus, and White Spot Syndrome Virus.

[0189] In some embodiments, when the sample is obtained from a patient, the patient has been34581226294Attorney Docket No.: 273089 / KBS-012WO / 577219diagnosed with, or is suspected to be suffering from an infection caused by a parasite selected from the group consisting of Plasmodium, Trypanosoma, Toxoplasma, Giardia, Leishmania, Cryptosporidium, helminthic parasites: Trichuris spp., Enterobius spp., Ascaris spp., Ancylostoma spp. and Necatro spp., Strongyloides spp., Dracunculus spp., Onchocerca spp. and Wuchereria spp., Taenia spp., Echinococcus spp. , and Diphyllobothrium spp., Fasciola spp., and Schistosoma spp.

[0190] In some embodiments, the sample is obtained from a patient diagnosed with, or suspected to be suffering from cancer. In some embodiments, the patient is being treated for cancer. In some embodiments, the patient has been treated for cancer. In some embodiments, the patient has been treated with one or more therapeutic agents, such as, but not limited to an inhibitory immune checkpoint blocker or inhibitor, a stimulatory immune checkpoint stimulator, agonist or activator, a chemotherapeutic agent, an anti-cancer agent, a radiotherapeutic agent, an anti-neoplastic agent, an anti-proliferation agent, an anti- angiogenic agent, an anti-inflammatory agent, an immunotherapeutic agent, a therapeutic antigen-binding molecule (mono- and multispecific antibodies and fragments thereof in any format (e.g., including without limitation DARTs®, Duobodies®, BiTEs®, BiKEs, TriKEs, XmAbs®, TandAbs®, scFvs, Fabs, Fab derivatives), bi-specific antibodies, non-immunoglobulin antibody mimetics (e.g., including without limitation adnectins, affibody molecules, affilins, affimers, affitins, alphabodies, anticalins, peptide aptamers, armadillo repeat proteins (ARMs), atrimers, avimers, designed ankyrin repeat proteins (DARPins®), fynomers, knottins, Kunitz domain peptides, monobodies, and nanoCLAMPs), antibody-drug conjugates (ADC), antibody-peptide conjugate), an oncolytic virus, a gene modifier or editor, a cell comprising a chimeric antigen receptor (CAR), e.g., including a T cell immunotherapeutic agent, an NK-cell immunotherapeutic agent, or a macrophage immunotherapeutic agent, a cell comprising an engineered T-cell receptor (TCR-T), or any combination thereof.

[0191] In some embodiments, the sample is a stool sample from a human donor.

[0192] In some embodiments, the microbial strain is a strain derived from a microbe selected from the group consisting of a bacterium, a synthetic bacterium, a synthetic organism, a fungus, a virus, an archaea, a parasite, and a genetically modified organism (e.g., wherein specific genes are added, deleted, mutated, driven to high expressions, and / or suppressed to low expression levels). In some embodiments, the at least one microbial strain is a bacterial strain. In some embodiments,35581226294Attorney Docket No.: 273089 / KBS-012WO / 577219the bacterial strain is a probiotic strain. In some embodiments, the at least one microbial strain is a genetically engineered strain. In some embodiments, the at least one microbial strain is a genetically engineered bacterial strain. In some embodiments, the genetically engineered strain is a genetically engineered bacterial strain. In a “genetically engineered’" strain, specific genes are added, deleted, mutated, driven to high expressions, and / or suppressed to low expression levels. The alterations are known to a person of ordinary skill in the art.EXAMPLES

[0193] EXAMPLE 1. rRNA Intensity and Regrowth Time

[0194] This experiment was designed to assess the validity of using rRNA intensity data as a metric to assess the difference in lag time between exponential and stationary phase bacteria of the same strain. At a high level, we aimed to assess if there were general trends in the intensity data that revealed ways to indicate if a population may regrow at a faster or slower rate when reinoculated in media. This in turn would help to give insight into the best formulation to use for drug process development.

[0195] To perform this experiment, stationary and exponential phase microbial cultures for a 10-strain community were prepared. Each of these 10 strains was individually cultured to a time point in the stationary phase and a timepoint in the exponential phase. Once the microbes reached the desired phase, a sample was removed from the culture and fixed. In total, there were 20 fixed microbial cultures, a stationary and an exponential phase culture for each of the 10 strains. Once samples were prepared a one-step FISH protocol assay was performed on all strains. The one step FISH protocol utilized a small encoding probe directly conjugated to a fluorescent readout probe. This probe was selected to minimize differences in intensities observed between species for technical hybridization or diffusion reasons rather than true differences in copy numbers of rRNA.

[0196] Additionally, on each slide processing the experimental samples, two control samples were included. The inclusion of these controls across all slides allowed for the normalization of intensity measurements to the intensity of the controls, thereby minimizing the effects of between assay differences, caused by environmental factors that are harder to control, but that could artificially influence the intensity of the samples. Once processed, the samples were imaged using confocal microscopy with a 561nm laser excitation. Multiple fields of view were imaged from each well, such that each sample (represented by one well) had a comparable number of microbes.36581226294Attorney Docket No.: 273089 / KBS-012WO / 577219Several hundred microbes was the lower limit of the amount imaged of each sample.

[0197] After collecting the data, the images were analyzed by measuring the intensity of each microbe in each sample by calculating the mean intensity of all pixels within the microbe at the peak channel for the fluorophore spectra. Once these mean intensities were calculated, the mean intensities for each sample were normalized with the in-assay control intensity, then plotted in a violin plot, with one plot per sample. By visualizing the intensity measurements in this way, the team was able to visualize whether there were distinct subpopulations that emerged within the samples, indicating heterogeneous intensities within a population. Additionally, the intensity measurements between the exponential and stationary phase samples of the same strain were compared to assess if the intensity measurements were different between the two phases. These intensity measurements and trends were then compared to regrowth data. The regrowth measurements were generated by inoculating new cultures with the exponential and stationary phase samples of each of the 10 strains, followed by quantifying the amount of time microbial cultures took to reanimate. Reanimation was measured by assessing the first signs of metabolic activity in the cultures.

[0198] Results from this initial experimentation revealed that the intensity measurements for microbes in the exponential phase were always seen to have greater than or equal to mean intensity measurements in the microbial population as compared to the stationary phase microbes. Additionally, all cultures inoculated from the exponential growth phase microbes reanimated in equal to or faster time than their paired stationary counterparts. Importantly, the strain which exhibited no significant difference in the exponential and stationary populations’ mean intensity also did not exhibit a difference in reanimation time between these two phases. Finally, it was observed that heterogeneous rRNA intensity within a population, characterized by two or more significant subpopulations in the plotted rRNA intensity graphs, may be an important trend influencing a microbial populations’ likelihood of reanimation.

[0199] As shown in Figure 1 and 2, the rRNA intensity is heterogenous across the population at any time point and for any strain. However, the mean of the population can change drastically between strains, time points, environments, etc.

[0200] As shown in Figure 1, there is a large a significant difference in the population measurement of rRNA intensity between the exponential and stationary phases. Similarly, there is a large difference in the reanimation time of the two populations when beginning from similar cell37581226294Attorney Docket No.: 273089 / KBS-012WO / 577219numbers.

[0201] As shown in Figure 2, there is no significant difference in the population measurement of rRNA intensity between the exponential and stationary phases. Similarly, there is no difference in the reanimation time of the two populations when beginning from similar cell numbers.

[0202] EXAMPLE 2. Synthetic Community

[0203] This experiment assessed the ability to measure individual strain’s viability accurately within a complex community. We aimed to address whether we could successfully detect the relative viability measurements of each species in a mixed community with a ground truth of what individual viability measurements we should detect in the community. Since other methods of detecting viability measurements do not have the ability to detect individual viability measurements in a mixed community, a synthetic community (a mixture of live and dead bacteria) was created to establish that we are able to correctly call a live or dead state in a community that uses complex HiPR-FISH barcodes.

[0204] A fixed stock of E. coll microbes was split in half. One half of the fixed microbes was not manipulated at all. This half acted as the “live” E. coli, as the microbes would not see any viability dye and should only stain with the DAPI dye. The second half was completely stained with viability dye and represented the “dead” E. coli. The same process was repeated with a second microbe, E.faecalis. Once these distinct conditions were created, each microbes’ “live” and “dead” community were mixed by volume to create a 50% “live” 50% “dead” community of E. coli and a 90% “live” 10% “dead” community of E. faecalis. Once the individual communities were created, each of the individual mixes were mixed together 1 : 1 to create a final community of 50% (by volume) total E. coli, with an individual viability of 50%, and 50% (by volume) total E.faecalis with a viability of 90%. Once the mixes were created, the HiPR-Vie assay was run, encoding with HiPR-FISH style specific encoding probes for E. coli and E. faecalis and stained with DAPI to establish a total cell count. After running the assay, the mix was imaged using 7 laser excitations with a confocal microscope. Several fields of view were imaged to sample a representative number of the microbial mix.

[0205] Once the data was collected, the images were analyzed to correctly classify the microbes in the pseudo community. In addition, the ratio between the DAPI and viability dyes present in each microbe was measured. Based on the measured presence of either dye, each microbe was marked as either “live” or “dead”. In the end, each microbe in the images was marked38581226294Attorney Docket No.: 273089 / KBS-012WO / 577219with a specific microbial identity, as well as a “live” or “dead” value. Once these values were established for all objects in the acquired field of views, the relative abundances of the microbes and their individual relative viability measurements were calculated. In the end, the assay and analysis called 53.6% E. faecalis and 46.4% E. coli in a representative field of view, as shown in Figure 3 and the table below. Additionally, the assay called 58% dead E. coli and 42% live E. coli in the field of view of the sample mix. Finally, 100% of the E. faecalis were marked as live in this field of view. These measurements confirmed the ability for this assay to integrate LIVE / DEAD® dye and corresponding measurements within HiPR-FISH classification without losing accuracy for either measurement metric.

[0206] Table A. Total Community Relative Abundance.Total Community Relative E.coli 46.4% E. faecalis 53.6% Abundance:

[0207] EXAMPLE 3. Testing Measurements on a Complex Microbial Community

[0208] In this experiment, we tested the measurement abilities on a more complex community, and on a real microbial culture, in which the viability will count true live and dead measurements. We tested the assay on a community that represented the complexity of a communities that we would be tested in practice. We also tested viability measurements on microbes that died from non-fixation causes.

[0209] A frozen aliquot of a complex community was treated with viability dye. Once treated according to the viability dye instructions, the stock was fixed for use with the HiPR-Vie assay. Then, the HiPR-Vie assay was run, encoding with HiPR-FISH style specific encoding probes for the drug substance community and stained with DAPI to establish a total cell count. After running the assay, the mix was imaged using 7 laser excitations with a confocal microscope. Several fields of view were imaged to sample a representative number of the microbial mix.

[0210] Once the data was collected, the images were analyzed to correctly classify the microbes in the drug substance community. In addition, the ratio between the DAPI and viability dyes present in each microbe was measured. Based on the measured presence of either dye, each39581226294Attorney Docket No.: 273089 / KBS-012WO / 577219microbe was marked as either “live” or “dead”. In the end, each microbe in the images was marked with a specific microbial identity, as well as a “live” or “dead” value. Once these values were established for all objects in the acquired field of views, the relative abundances of the microbes and their individual relative viability measurements were calculated. For this sample, there was no ground truth data for the viability measurements, so reports instead demonstrated the platform’s ability to both correctly identify individual microbes in the community, as well as distinguish if those microbes were alive or dead.

[0211] Figure 4 shows classified microbiota in a complex synthetic community (on the left), following the application of viability measurements, dead microbiota were removed from classification (on the right).

[0212] Figure 5 shows the frequency of live and dead cells are enumerated for several strains shown in Figure 4.

[0213] EXAMPLE 4. VIABILITY MEASUREMENTS ON FRESHLY ISOLATED STOOL DISTINGUISH LIVE AND DEAD CELLS

[0214] A fresh canine stool sample was isolated and embedded in OCT in a liquid nitrogen bath. The stool sample was sectioned into 10-micron sections in a cryotome at -20°C and applied to microscope slides. Viability dye was added to the slides directly in an anaerobic chamber for 30 minutes at room temperature. The slide was rinsed three times with IxPBS in the anaerobic chambers, and fixed in 4% formaldehyde for 15 minutes at room temperature. The fixative was removed and the sample was washed. HiPR-FISH was carried out using a fluorescent probe targeting the region of a 16S ribosomal RNA. DAPI-stain was added to identify DNA. The sample was mounted and imaged on a Zeiss i980 Axi oVert Microscope using 405 nm and 561 nm laser lines. Imaging data was then processed.

[0215] EXAMPLE 5. VIABILITY MEASUREMENTS ON CELLS EXPOSED TO HEAT OR OXYGEN

[0216] A lyophilized complex community was resuspended in media, and immediately exposed to heat (60°C) or placed in a 50 mL tube and rotated under atmospheric conditions. Cells were then stained with viability dye and assayed to determine rRNA (biomarker) intensity.

[0217] As shown in Figure 7 the viability measurements sensitively and accurately determine the cell state. As shown in Figure 8, molecular biomarkers can be used to understand the health of a cell, taxon, or population.40581226294Attorney Docket No.: 273089 / KBS-012WO / 577219

[0218] Details of one or more embodiments are set forth in the accompanying drawings and description. Other features, objects, and advantages will be apparent from the description, drawings, and claims. Although a number of embodiments of the invention have been described, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. It should also be understood that the appended drawings are not necessarily to scale, presenting a somewhat simplified representation of various features and basic principles of the invention.41581226294

Claims

Attorney Docket No.: 273089 / KBS-012WO / 577219WHAT IS CLAIMED IS:

1. A method of detecting viable cells in a sample, the method comprising:(a) staining the cells with a fixable viability dye;(b) washing away the fixable viability dye;(c) fixing the cells of the sample on an array;(d) performing an identifying assay on the cells;(e) adding a nuclear dye to the cells; and(f) mounting and imaging the cells with a microscope to assess the viability of the cells.

2. The method of claim 1, wherein the imaging with a microscope produces a first, second, and third detectable signal,wherein the first detectable signal derives from the viability dye and is indicative of the presence of dead cells;wherein the second detectable signal derives from the nuclear dye and is indicative of the presence of a viable cell fraction; andwherein the third detectable signal derives from the identifying assay and is indicative of a known microbial strain.

3. The method of claim 1 or claim 2, wherein the identifying assay comprises:(A) providing a first set of probes,wherein the first set of probes comprises at least one first encoding probe and at least one first emissive readout probe; and(B) contacting the first set of probes with the fixed cells to form a first complex; wherein the at least one first emissive readout probe emits a first color, and wherein the first color is assigned to a known microbial strain.

4. The method of claim 3, wherein the imaging acquires one emission spectra from the at least one first emissive readout probe.42581226294Attorney Docket No.: 273089 / KBS-012WO / 5772195. The method of any one of claims 1-4, wherein the viability of cells is assessed via a relative signal analysis or a barcode classification.

6. The method of claim 5, wherein the relative signal analysis measures the ratio between the amount of viability dye signal relative to the amount of nuclear dye signal.

7. The method of claim 5, wherein the barcode classification assigns a bit to the viability dye, wherein the bit “1” is assigned to dead cells and bit “0” is assigned to live cells.

8. The method of any one of claims 1-7, wherein the method further comprises (e.l) incorporating controls into the sample prior to imaging, wherein the controls comprise community-based controls or strain-specific controls.

9. The method of claim 8, wherein the community-based control comprises a preparation of “live” and “dead” controls, wherein the “live” control comprises a portion of the sample that is fixed and washed and has not been exposed to a viability dye; and wherein the “dead” control comprises a portion of the sample that is fixed and washed and has been exposed to a viability dye.

10. The method of claim 8, wherein the strain-specific control comprises a preparation of “live” and “dead” pure culture of a known, individual strain, wherein the “live” pure culture comprises a portion of a culture of a known, individual strain that is fixed and washed and has not been exposed to a viability dye; and wherein the “dead” pure culture comprises a portion of a culture of a known, individual strain that is fixed and washed and has been exposed to a viability dye.

11. The method of any one of claims 1-10, wherein the method further comprises (e.2) performing a second identifying assay, wherein the second identifying assay produces a fourth detectable signal.43581226294Attorney Docket No.: 273089 / KBS-012WO / 57721912. The method of claim 11, wherein the fourth detectable signal derives from the second identifying assay and is indicative of a known gene, a known protein, or a known marker of cell stress or cell viability.

13. The method of claim 11 or claim 12, wherein the second identifying assay is indicative of a known gene, and wherein the second identifying assay comprises:contacting at least one second encoding probe with the cell to produce a second complex, wherein each encoding probe comprises an mRNA targeting sequence and an initiator sequence;adding two different DNA amplifier sequences to the second complex to produce a third complex, wherein each DNA amplifier comprises an initiator complimentary sequence and a readout sequence; andadding two second emissive readout probes to the third complex, wherein each second emissive readout probe comprises a fluorophore and a complimentary sequence to the second readout sequence of a corresponding DNA amplifier sequence.

14. The method of claim 11 or claim 12, the second identifying assay is indicative of a known protein, and wherein the second identifying assay comprises: identifying the presence of immunoglobulins on the surface of the cell, wherein the identifying the presence of immunoglobulins on the surface of a cell comprises incorporating an oligonucleotide-conjugated antibody or a fluorophore conjugated antibody.

15. The method of any one of claims 1-14, wherein the method further comprises (g) assessing the morphology of the cells.

16. The method of claim 15, wherein the morphology assessment comprises assessing the area, volume, specific radius, Euler number, solidity, eccentricity, perimeter, convex area, orientation relative to neighbors, geometry, or shape of the cell.

17. The method of claim 15 or claim 16, wherein the method further comprises (g.1.1) comparing the morphology assessments with reference data of known cells in a natural or healthy states to assess the health of the cells.44581226294Attorney Docket No.: 273089 / KBS-012WO / 57721918. The method of claim 15 or claim 16, wherein the method further comprises (g.1.2) comparing the morphology assessments with reference data of known cells in an unnatural, stressed, diseased, or dead state to assess the health of the cells.

19. The method of any one of claims 1-18, wherein the viability dye comprises LIVE / DEAD® Fixable Blue stain, LIVE / DEAD® Fixable Violet stain, LIVE / DEAD® Fixable Lime stain, LIVE / DEAD® Fixable Aqua stain, LIVE / DEAD® Fixable Yellow stain, LIVE / DEAD® Fixable Green stain, LIVE / DEAD® Fixable Olive stain, LIVE / DEAD® Fixable Orange stain, LIVE / DEAD® Fixable Red stain, LIVE / DEAD® Fixable Far Red stain, LIVE / DEAD® Fixable Scarlet stain, LIVE / DEAD® Fixable Near-IR (775) stain, LIVE / DEAD® Fixable Near-IR (780) stain, LIVE / DEAD® Fixable Near 1R (876) stain, or 5-Cyano-2, 3 -ditolyl tetrazolium chloride (CTC).

20. The method of any one of claims 1-19, wherein the nuclear dye comprises 4', 6-diamidino-2-phenylindole (DAPI).

21. The method of any one of claims 1-20, wherein the sample comprises at least one cell, a cell suspension, a tissue biopsy, a tissue specimen, bone biopsies, organoids, three-dimensional hydrogel scaffolds, transwell systems, or plant biopsies.

22. The method of any one of claims 1-21, wherein the sample is a cell.

23. The method of any one of claims 1-22, wherein the cell is a bacterial or eukaryotic cell.

24. The method of any one of claims 1-23, wherein the sample is a stool sample from a human donor.

25. The method of any one of claims 1-24, wherein the microbial strain is a strain derived from a microbe selected from the group consisting of a bacterium, a synthetic bacterium, a synthetic organism, a fungus, a virus, an archaea, a parasite, and a genetically modified organism.45581226294Attorney Docket No.: 273089 / KBS-012WO / 57721926. The method of claim 25, wherein the microbial strain is a bacterial strain.

27. The method of any one of claims 1-26, wherein the microbial strain is a genetically engineered strain.

28. The method of claim 27, wherein the at least one microbial strain is a genetically engineered microbial strain.

29. The method of any one of claims 3-28, wherein each of the encoding probes comprise a targeting sequence.

30. The method of claim 29, wherein the targeting sequence targets at least one of messenger RNA (mRNA), micro RNA (miRNA), long non-coding RNA (IncRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), transfer RNA (tRNA), Crispr RNA (crRNA), transactivating crispr RNA (tracrRNA), mitochondria RNA, Intronic RNA, viral mRNA, viral genomic RNA, environmental RNA, double-stranded RNA (dsRNA), small nuclear RNA (snRNA), small nucleolar (snoRNA), piwi-interacting RNA (piRNA), genomic DNA, synthetic DNA, DNA, plasmid DNA, a plasmid, viral DNA, retroviral DNA, environmental DNA, extracellular DNA, a protein, a small molecule, or an antigenic target.

31. The method of claim 30, wherein the targeting sequence targets mRNA and / or rRNA.

32. The method of any one of claims 29-31, wherein the targeting sequence targets a 16S rRNA sequence, a 5S rRNA sequence, and / or a 23S rRNA sequence in the microbial strain.

33. The method of any one of claims 29-31, wherein the targeting sequence targets a 18S rRNA sequence, a 5.8S rRNA sequence, and / or a 28S rRNA sequence in the in the microbial strain, wherein the microbial strain is a eukaryotic microbial strain.

34. The method of any one of claims 2-33, wherein the emissive readout probe comprises a label on the 5’ and / or 3’ end.46581226294Attorney Docket No.: 273089 / KBS-012WO / 57721935. The method of claim 34, wherein the label is Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 561, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 647-R-phycoerythrin, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 680-allophycocyanin, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, Alexa Fluor Plus 405, Alexa Fluor Plus 488, Alexa Fluor Plus 555, Alexa Fluor Plus 594, Alexa Fluor Plus 647, Alexa Fluor Plus 680, Alexa Fluor Plus 750, Alexa Fluor Plus 800, Pacific Blue, Pacific Green, Rhodamine Red X, DyLight 485-LS, DyLight-510-LS, DyLight 515-LS, DyLight 521-LS, Hydroxy coumarin, methoxy coumarin, Cy2, FAM, Fluorescein FITC, R-phycoerythrin (PE), Tamara, Cy3.5 581 , Rox, Red 613, Texas Red, Cy5, Cy5.5, Cy7, Allophycocyanin, ATTO 430LS, ATTO 490LS, ATTO 390, ATTO 425, Cyan 500 NHS-Ester, ATTO 465, ATTO 488, ATTO 495, ATTO RhollO, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rhol 1, ATTO Rhol2, ATTO Thiol2, ATTO RholOl, ATTO 590, ATTO 594, ATTO Rhol3, ATTO 610, ATTO 620, ATTO Rhol4, ATTO 633, ATTO 643, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxal2, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740.

36. The method of any one of claims 1-35, wherein the microscope is a hyperspectral microscope, widefield epifluorescence microscope, widefield microscope, whole-slide scanner, point scanning confocal microscope, spinning disk confocal microscope, lattice lightsheet microscope, or light field microscope.

37. The method of claim 36, wherein the detection strategy used is channel, spectral, channel and fluorescence lifetime, or spectral and fluorescence lifetime.

38. The method of any one of claims 1-37, wherein the array is a microscope slide, at least one chamber, at least one microfluidic device, at least one well, at least one plate, at least one filter membrane, or at least one adhesive tape.47581226294

Images