Anchored bridge nucleic acid probes and methods thereof; ribonuclease-insensitive methods for determining cellular nucleic acid in a biolofical sample

Abstract

The present disclosure is generally directed to anchoring bridge nucleic acid probes in a polymer matrix, which are configured to hybridize to cellular nucleic acid, embedding in a polymer matrix, clearing cellular components including the cellular nucleic acid, contacting the anchored bridge probes with primary probes that hybridize with the bridge probes and imaging. The disclosure describes methods for imaging cellular RNA in an RNase insensitive method.

Description

VIZG019.PCT / USANCHORED BRIDGE NUCLEIC ACID PROBES AND METHODS THEREOF; RIBONUCLEASE-INSENSITIVE METHODS FOR DETERMING CELLULAR NUCLEIC ACID IN A BIOLOGICAL SAMPLE RELATED APPLICATIONS

[0001] This application claims priority to U. S. Ser. No. 63 / 760,875 filed on February 20, 2025, which is incorporated by reference into this application in its entirety.FIELD OF THE DISCLOSURE

[0002] This application relates generally to the field of in situ imaging, and in particular, relates to methods of determining nucleic acid targets within biological samples.

[0003] BACKGROUND OF THE DISCLOSURE

[0004] To accurately profile the gene expression in biological samples in situ, a spatial transcnptomics technique with high detection efficiency and single molecule resolution is required. In situ single cell transcriptomic imaging technology, such as Multiplexed Error-Robust Fluorescence In Situ Hybridization (MERFISH), enables the direct profiling of the spatial organization of intact tissue with subcellular resolution. Through combinatorial labeling and sequential imaging, difterent RNA species are imaged by fluorescence microscopy to generate a specific pattern of fluorescent ON and OFF signal, an optical barcode that can be used to resolve the location and quantity of different RNA species. This spatially resolved RNA-profiling data gives a physical picture for the cell or tissue of interest, which can help elucidate the intricate interplay between different cell types in complex biological systems.

[0005] However, RNA species are vulnerable to chemical insults from endogenous and exogenous sources, and the ubiquitous presence of ribonuclease (RNase) in the environment poses a great challenge to RNA imaging. This challenge is particularly concerning for RNA imaging technologies, such as MERFISH, that require prolonged imaging time up to several days. Reagents such as RNase inhibitors can be used to prevent RNA degradation. However, chemical RNase inhibitors are costly, and they need to be replenished during RNA imaging as their activity drops overtime, while vanadyl ribonucleoside complex (VRC) is not compatible with the imaging process for MERFISH. Therefore, there is a strong need to develop a RNase-insensitive methods to analyze the RNA in biological samples.VIZG019.PCT / US

[0006] SUMMARY OF THE DISCLOSURE

[0007] Described herein are methods and reagents thereof for in situ single-cell transcriptomic analysis from biological samples.

[0008] In embodiments provided herein are methods for anchoring within a polymer matrix a complementary target of a distinct nucleic acid species and clearing cellular components. In embodiments, the methods comprise contacting a biological sample comprising the distinct nucleic acid species with a bridge probe and an anchoring agent, wherein the bridge probe comprises an acrydite moiety and hybridizes with the distinct nucleic acid species and the anchoring agent forms a covalent bond with the bridge probe, wherein when hybridized the bridge probe provides a complementary target of the distinct nucleic acid species; embedding the biological sample in a polymer matrix wherein the complementary target of the distinct nucleic acid species and the anchoring agent each form a covalent bond with the polymer matrix; and clearing cellular components from the polymer matrix wherein the complementary target of the distinct nucleic acid species remain anchored in the polymer matrix to form a matrix anchored complementary target sample. It is understood that the complementary target is the anchored bridge probes. In certain embodiments, the methods further comprise imaging the complementary target of the distinct nucleic acid species. In embodiments, the methods comprise contacting the anchored bridge probes with a plurality of primary nucleic acid probe pools each of the primary nucleic acid probes comprising a bridge probe target sequence and one or more read sequences, wherein each pool of primary nucleic acid probes hybridize to a distinct complementary target of the distinct RNA species and each pool of primary probes encode a N-bit code that was assigned to each distinct RNA species, wherein each assigned N-bit code is a valid codeword with a Hamming distance of 2 or more between valid codewords; contacting the bound primary nucleic acid probes with a plurality of fluorescent readout probes that hybridize to the read sequences of the primary nucleic acid probes; imaging the readout probes bound to the primary nucleic acid probes; and, repeating steps c) and f) in one or more sequential hybridization and imaging rounds until all N positions in the N- bit code have been imaged providing an imaged bit code corresponding to each of the distinct nucleic acid species in a spatial organization

[0009] In certain embodiments provided herein are methods for imaging a complementary target of a distinct nucleic acid species and clearing cellular components. In embodiments, the methods comprise contacting a biological sample comprising the distinct nucleic acid species with a bridge probe and an anchoring agent, wherein the bridge probe comprises an acrydite moiety and hybridizesVIZG019.PCT / USwith the distinct nucleic acid species and the anchoring agent forms a covalent bond with the bridge probe, wherein when hybridized the bridge probe provides a complementary target of the distinct nucleic acid species; embedding the biological sample in a polymer matrix wherein the complementary target of the distinct nucleic acid species and the anchoring agent each form a covalent bond with the polymer matrix; clearing cellular components from the polymer matrix wherein the complementary target of the distinct nucleic acid species remain anchored in the polymer matrix to form a matrix anchored complementary target sample; contacting the anchored complementary target with a primary nucleic acid probe comprising a bridge probe hybridization sequence and one or more read sequences; and, contacting the primary nucleic acid probe with a plurality of fluorescent readout probes that hybridize to the read sequences of the primary nucleic acid probes and imaging the complementary target of the distinct nucleic acid species.

[0010] In certain other embodiments provided herein, are methods for generating and anchoring within a polymer matrix a complementary target of a plurality of distinct nucleic acid species. In embodiments the method comprises contacting a sample comprising a plurality of distinct nucleic acid species with a plurality of nucleic acid bridge probe pools each of the bridge probes comprising a target sequence and a moiety that covalently binds to a polymer matrix, wherein when hybridized the bridge probe pools provide a complementary target of the distinct nucleic acid species; polymerizing the polymer matrix within the sample; anchoring the complementary target of the distinct nucleic acid species to the polymer matrix wherein the bridge probes covalently bind the polymer matrix; and, clearing cellular components trom the polymer matrix wherein the complementary target of the distinct nucleic acid species remain anchored in the polymer matrix. In certain embodiments, the methods further comprise contacting the bridge probes with a plurality of primary nucleic acid probe pools each of the primary nucleic acid probes comprising a bridge probe target sequence and one or more read sequences, wherein each pool of nucleic acid probes hybridize to a distinct complementary target of the distinct nucleic acid species and each pool of probes encode a N-bit code that was assigned to each distinct nucleic acid species; contacting the bound primary nucleic acid probes with a plurality of fluorescent readout probes that hybridize to the read sequences of the primary nucleic acid probes; imaging the readout probes bound to the primary nucleic acid probes; and, repeating steps f) and g) in one or more sequential hybridization and imaging rounds until all N positions in the N- bit code have been imaged providing an imaged bit code corresponding to each of the distinct nucleic acid species in a spatial organization.VIZG019.PCT / US

[0011] Also provided herein are bridge nucleic acid probe comprising an acrydite moiety and a target sequence configured to hybridize with a distinct nucleic acid species in a biological sample. In certain embodiments provided herein is a plurality of bridge nucleic acid probe pools each of the bridge probes comprising: a target sequence and an acrydite moiety, wherein each pool of bridge probes is configured to hybridize to a distinct RNA species in a sample.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 shows the workflow of Protocol A (methods of this disclosure) and Protocol B (methods of this disclosure). See Example 2.

[0013] Figure 2 shows generation of a bridge probe pool comprising a 5’ acrydite moiety. See Example 1.

[0014] Figure 3 shows a bar graph with transcripts per cell (“MERFISH counts”) in U2OS cells using anchored bridge probes (“Bridge TRRC library”) compared to unanchored probes (“Unmodified library”) with and without addition of an anchoring agent (“Anchoring Agent 1”). See Example 3.

[0015] Figure 4 shows correlation data with bulk RNAseq for the unanchored probes (“Ctrl”) and anchored bridge probes (“Bridge probe pools”) and correlation between two datasets (with and without the anchoring agent) from the data of Figure 3. See Example 3.

[0016] Figure 5 shows spatial distribution of those imaged gene transcripts from U2OS cells. See Example 3.

[0017] Figure 6 shows (Fig 6A) a bar graph with transcripts per field of view (“MERFISH counts”) in a mouse brain tissue sample using anchored bridge probes (“Bridge probe mediated modification”) compared to no bridge probes (“Control”); (Fig 6B) correlation data of the transcripts per field of view (“MERFISH counts”) with bulk RNAseq using the anchored bridge probe (“bridge probe”) and correlation data between the two different protocols (anchored bridge probes and no bridge probes); and (Fig 6C) spatial distribution of four imaged gene transcripts in the mouse brain tissue sample. See Example 5.

[0018] Figure 7 shows a bar graph with transcripts per cell (“MERFISH counts”) in fresh frozen mouse heart tissue (A) and FFPE mouse liver tissue (B) using anchored bridge probes (“Bridge probe mediated modification”) compared to no bridge probes (“Ctrl”). See Example 6.VIZG019.PCT / USDETAILED DESCRIPTION OF THE DISCLOSURE

[0019] The present disclosure generally relates to anchoring bridge nucleic acid probes in a biological sample that is embedded in a polymer matrix. The bridge probes of this disclosure are designed and generated to hybridize with a cellular nucleic acid target(s) and also contain a moiety that can form a covalent bond with a polymer matrix (e.g., 5’ acrydite moiety). Once immobilized, downstream steps may include, clearing the cellular components, removing the cellular nucleic acid target, adding primary probes that are designed and generated to hybridize with the anchored bridge probes, and then imaging via a secondary labeled probe. Immobilizing or covalently binding the bridge nucleic acid probes in the polymer matrix embedded biological sample provides an RNase-insensitive method for imaging cellular nucleic acid. In embodiments, the bridge probes comprise a target sequence that hybridizes to a cellular nucleic acid target and a moiety that forms a covalent bond with a polymer matrix. In embodiments, the moiety that forms a covalent bond with a polymer matrix comprises an acrydite moiety, an amine moiety or an allyl moiety. One of skill in the art understands the moiety is selected based on preferred chemistry and the selection of the polymer matrix (e.g., polyacrylamide gel). Derivatizing chemical compounds or oligonucleotides with the desired reactive group is well understood by one of skill in the art. In embodiments, the biological sample is a cell suspension, a cell from cell culture, fresh or frozen tissue samples, or FFPE (formalin fixed paraffin embedded) tissue samples. The present disclosure also provides preparation of tissue samples that allows in situ single-cell trans criptomic imaging (e.g., MERFISH, smFISH) to detect nucleic acid targets in the samples. The methods ot the present disclosure can be used for the preparation ot gene expression profiles of tissue samples. Other aspects are generally directed to systems or kits involving such methods or the like.

[0020] Methods of this disclosure comprise a method to determine the nucleic acids in biological samples resistant to RNase degradation by utilizing oligo probes (“bridge nucleic acid probes”) containing target regions that can hybridize to nucleic acids in a cell or tissue, a moiety that forms a covalent bond with a polymer matrix (e.g., 5’ labeled acrydite moiety). See Figure 2 and Example 1. These bridge nucleic acid probes comprising an acrydite moiety are added to a biological sample where they hybridize to cellular nucleic acid target(s), the biological sample is embedded with a polymer matrix wherein the acrydite moiety of the bridge probe covalently binds with the polymer matrix. Once the bridge probes are anchored or immobilized in the polymer matrix, cellular components, including the cellular nucleic acid target hybridized to the bridge probes, can be cleared and removedVIZG019.PCT / USfrom the sample. Removal of the cellular nucleic acid bound to the bridge probe facilitates hybdnzation of the primary nucleic acid probes which comprise a bridge probe target sequence and one or more read sequences. Subsequently secondary probes and / or labels can be added and imaging completed. See Example 3. In certain embodiments, an anchoring agent is added to the bridge probe in situ, wherein the anchoring agent forms a covalently bond with the bridge probe and then with the polymer matrix when the biological sample is embedded with the polymer matrix. See Example 2, Figure 1 (“Protocol A”) and Figure 3. In certain other embodiments, when fragmentation of the nucleic acid, especially mRNA, is suspected (e.g., FFPE samples), the cellular nucleic acid is anchored in a polymer matrix before the bridge probes comprising a moiety that forms a covalent bond with the polymer matrix. See Example 6, Figure 1 (“Protocol B”) and Figure 7. In embodiments, anchoring the cellular nucleic acid target comprises contacting the biological sample with a first and / or second anchoring agent, wherein the first anchoring agent forms a covalent bond with the target nucleic acid and the second anchoring agent comprises an oligonucleotide that hybridizes with the target nucleic acid.

[0021] In embodiments, the biological sample may be immobilized or embedded within a polymer or a gel, partially or completely. In some cases, the sample may be embedded within a relatively large polymer or gel, which can then be sectioned or sliced in some cases to produce smaller portions for analysis, e.g., using various microtomy techniques commonly available to those of ordinary skill in the art. For instance, tissues or organs may be immobilized within a suitable polymer or gel. In embodiments, the polymer may be selected to be relatively optically transparent. The polymer may also be one that does not significantly distort during the polymerization process, although in some cases, the polymer may exhibit some distortion. In some cases, the amount of distortion may be determined as a relative change in size that is less than 5, less than 4, less than 3, less than 2, less than 1.5, less than 1.3, or less than 1.2 (i.e., a change in size of 2 means that a sample doubles in linear dimension), or inverses of these (i.e., an inverse change in size of 2 means that a sample halves in linear dimensions).

[0022] Examples of suitable polymers include polyacrylamide and agarose. In some cases, the polymer is a gel or a hydrogel. A variety of polymers could be used in various embodiments that involve chemical cross links between gel subunits, including but not limited to acrylic acid, acrylamide, ethylene glycol diacrylate, ethylene glycol dimethacrylate, polyethylene glycol dimethacrylate); and / or hydrophobic or hydrogen bonding interactions, such as poly(N-isopropyl acrylamide), methylVIZG019.PCT / UScellulose, (ethylene oxide)-(propylene oxide)-(ethylene oxide terpolymers, sodium alginate, poly(vinyl alcohol), alginate, chitosan, gum Arabic, gelatin, and agarose.

[0023] In certain embodiments are methods for anchoring a bridge nucleic acid probe within a matrix and clearing cellular components, which can then be used for downstream imaging of the nucleic acid targets. The bridge probes form a copy of the cellular nucleic acid that can then be hybridized with a primary nucleic acid probe. In embodiments, the methods comprise contacting biological sample with a bridge nucleic acid probe and an anchoring agent, wherein the bridge probe comprises an acrydite moiety and hybridizes with a target nucleic acid and the anchoring agent forms a covalent bond with the primary nucleic acid probe; embedding the biological sample in a polymer matrix wherein the bridge nucleic acid probe and the anchoring agent each form a covalent bond with the polymer matrix; and clearing cellular components from the polymer matrix wherein the bridge nucleic acid probe hybridized to the target nucleic acid remains anchored in the polymer matrix to form a matrix anchored bridge nucleic acid probe sample.

[0024] In certain other embodiments, the methods compnse contacting a biological sample comprising the distinct nucleic acid species with a bridge probe and an anchoring agent, wherein the bridge probe comprises an acrydite moiety and hybridizes with the distinct nucleic acid species and the anchoring agent forms a covalent bond with the bridge probe, wherein when hybridized the bridge probe provides a complementary target of the distinct nucleic acid species; embedding the biological sample in a polymer matrix wherein the complementary target of the distinct nucleic acid species and the anchoring agent each form a covalent bond with the polymer matrix; and clearing cellular components from the polymer matrix wherein the complementary target of the distinct nucleic acid species remain anchored in the polymer matrix to form a matrix anchored complementary target sample. In embodiments, the method further comprises contacting the anchored bridge probes with a plurality of primary nucleic acid probe pools, each of the primary nucleic acid probes comprising a bridge probe target sequence and one or more read sequences, wherein each pool of primary nucleic acid probes hybridize to a distinct complementary target of the distinct RNA species and each pool of primary probes encode a N-bit code that was assigned to each distinct RNA species, wherein each assigned N-bit code is a valid codeword with a Hamming distance of 2 or more between valid codewords. In embodiments, the method further comprises imaging the hybridized primary nucleic acid probes. In certain embodiments, the methods comprise contacting the primary nucleic acid probes with a plurality of fluorescent readout probes that hybridize to the read sequences of theVIZG019.PCT / USprimary nucleic probes; imaging the readout probes bound to the primary nucleic acid probes; and, repeating steps a) and b) in one or more sequential hybridization and imaging rounds until all N positions in the code have been imaged providing an imaged code corresponding to each distinct nucleic acid species in a spatial organization.

[0025] In certain embodiments, the target nucleic acid is RNA, in particular mRNA, wherein the methods comprise contacting a biological sample suspected of containing fragments RNA (e.g., formalin fixed paraffin embedded (FFPE) tissue sample) with a first and / or second anchoring agents, wherein the first anchoring agent comprises an alkylating agent that forms a covalent bond with the target nucleic acid and the second anchoring agent comprises a polyT sequence that is complementary and hybridizes to the target RNA and wherein the first anchoring agent and the second anchoring agent each comprise an acrydite moiety that covalently binds to the matrix; embedding the biological sample in a polymer matrix wherein the first and second anchoring agents each form a covalent bond with the polymer matrix; and, clearing the cellular components from the polymer matrix wherein the target RNA remains anchored in the polymer matrix. The bridge probes of this disclosure are then added to the anchored RNA and a second gel embedding step is performed to anchor the bridge probe to the polymer gel rendering the imaging RNase insensitive. This step is then followed by addition of the primary probes and imaging steps.

[0026] In certain embodiments, FFPE tissue sections are prepared for transcriptome analysis (e.g., RNA transcripts) comprising the steps of deparaffinization, ethanol rehydration, antigen retrieval, followed by the addition of a first and / or second anchoring agents (e.g., such as two functionally different or distinct anchoring agents) to the tissue sample, wherein a first anchoring agent forms a covalent bond with the target nucleic acid and a second anchoring agent (also referred to herein as an “anchor probe”) comprises an oligonucleotide that hybridizes with the target nucleic acid. In this way, the target nucleic acid has been functionalized with two separate anchoring agents to form an anchor treated tissue sample. This anchoring treatment step improves immobilization of the target nucleic acid within a gel or polymer matrix, wherein mRNA, in particular, can become fragmented during the FFPE process of fixing tissue sections. Each of these anchoring agents further comprise a chemical moiety (e.g., reactive group) that can form a covalent bond with a polymer matrix either during (polymerization) or after the polymer matrix has formed. Accordingly, after the first nad / or second anchoring agents are added to the tissue sample the sample is then embedded in a polymer matrix wherein the first and second anchoring agents each form a covalent bond with the polymer matrix.VIZG019.PCT / USIn this way, the target nucleic acids are immobilized in the polymer gel matrix. In certain embodiments, the polymer matrix is a polyacrylamide gel and the first and second anchoring agents each comprise a reactive group that will form a covalent bond with acrylamide (e.g., acrydite).

[0027] After the gel embedding step, the cellular components (also referred to herein as a tissue clearing or digestion step) are removed using reagents and methods known in the art (e.g., protease digestion). In embodiments, this clearing step, in combination with the use of a first and / or second anchoring agents when starting with FFPE tissue sample, removes the protein crosslinking induced by the formalin fixing process exposing target nucleic acid to a complimentary bridge oligonucleotide probe designed to hybridize to a target sequence in the anchored nucleic acid.

[0028] In embodiments, provided herein are methods for imaging a nucleic acid target (or a complementary target thereof) within a matrix and clearing cellular components. The method comprises contacting a biological sample comprising the distinct nucleic acid species with a bridge probe and an anchoring agent, wherein the bridge probe comprises an acrydite moiety and hybridizes with the distinct nucleic acid species and the anchoring agent forms a covalent bond with the bridge probe, wherein when hybridized the bridge probe provides a complementary target of the distinct nucleic acid species; embedding the biological sample in a polymer matrix wherein the complementary target of the distinct nucleic acid species and the anchoring agent each form a covalent bond with the polymer matrix; clearing cellular components from the polymer matrix wherein the complementary target of the distinct nucleic acid species remain anchored in the polymer matrix to form a matrix anchored complementary target sample; contacting the anchored complementary target with a primary nucleic acid probe comprising a bridge probe hybridization sequence and one or more read sequences; and, contacting the primary nucleic acid probe with a plurality of fluorescent readout probes that hybridize to the read sequences of the primary nucleic acid probes and imaging the complementary target of the distinct nucleic acid species. In certain embodiments the methods include contacting nucleic acid targets (e.g., RNA transcripts) with a first and / or second anchoring agent to enhance immobilization of RNA transcript efficiency before polymer matrix embedding and addition of the modified bridge probes of this disclosure. In embodiments, the methods include tissue clearing (e.g., removing non-target cellular components) prior to contacting the sample with acrydite modified bridge nucleic acid probes (e.g., MERFISH probes, smFISH probes, etc.) to enhance the efficiency of bridge probe binding by exposing target nucleic acid after crosslinked proteins have been cleared. Because the acrydite moiety of the bridge probe needs to form a covalent bond with the polymerVIZG019.PCT / USmatrix during polymerization, a second polymer matrix is added to the sample. This renders the sample RNase insensitive, and RNase can optionally be added to the sample removing the nucleic acid target and leaving the anchored bridge probes which are subsequently imaged in situ as a proxy for the cellular nucleic acid target. In embodiments, RNase treatment of the sample containing the anchored bridge probe improves binding of the primary nucleic acid probes. In certain embodiments, the methods comprise contacting the RNA transcripts (e.g., nucleic acid target) with a first and / or second anchoring agents wherein a first anchoring agent forms a covalent bond with the target nucleic acid, and the second anchoring agent (anchor probe) comprises an oligonucleotide that hybridizes with the target nucleic acid, embedding the tissue sample comprising the first and / or second anchoring agents in a gel matrix whereby the RNA transcripts are immobilized in the polymer gel matrix when the first and second anchoring agents each form a covalent bond with the polymer matrix, digesting or clearing the tissue and / or non-target cellular components followed by contacting the immobilized nucleic acid (e.g., RNA transcripts) with a plurality of acrydite modified bridge probes that specifically hybridize to the immobilized target nucleic acid, adding a second polymer matrix, hybridizing with primary probes and imaging the immobilized bridge probe / primary probe hybridized complex.

[0029] The primary probes may be, for example, MERFISH probes or smFISH probes, and may be substantially complementary to the bridge probes, which are complimentary to mRNA or other RNAs, for example, for transcriptome analyses. The primary oligonucleotide probes may also include signaling entities, e.g., fluorescent signaling entities, for imaging and / or analysis of the sample. In certain embodiments, a secondary oligonucleotide probe that hybridizes to the primary probe comprises an imaging moiety (e.g., fluorescent signaling entities), wherein imaging comprises adding one or more of the secondary probes. In some embodiments, die mediod further comprises creating codewords or barcodes based on a distribution of the bound nucleic acid probes within the sample. In embodiments, the methods comprise contacting the biological sample comprising a plurality of distinct RNA species with a plurality of bridge nucleic acid probe pools and then a complementary plurality of primary nucleic acid probe pools each of the nucleic acid probes comprising a target sequence, one or more read sequences, wherein each pool of bridge nucleic acid probes hybridize to a distinct RNA species, and then each pool of primary probes hybridize to a corresponding pool of bridge probes and each pool of primary probes encode a N-bit code that was assigned to each distinct RNA species, wherein each assigned N-bit code is a valid codeword with a Hamming distance of 2 or more between valid codewords. The read sequences corresponded to a bit value of 1, wherein eachVIZG019.PCT / USsubsequent round of imaging produces a 0 (when no labeled readout probe binds a read sequence of the primary probe pool) or a 1 (when a readout probe binds or hybridizes to a read sequence) and in this way an N-bit codeword is decoded when all rounds of imaging are complete.

[0030] In embodiments, the method comprises contacting the primary nucleic acid probes with a plurality of readout probes comprising a fluorescent label, wherein the readout probes hybridize to the read sequences of the primary nucleic acid probes; imaging the readout probes bound to the primary nucleic acid probes; and, repeating steps in one or more sequential hybridization and imaging rounds until all N positions in the N-bit code have been imaged providing an imaged codeword corresponding to each distinct RNA species in a spatial organization. In embodiments, the valid codewords have a Hamming distance equal to or greater than 4 between each of the valid codewords. In this way, an error (e.g., a 0 incorrectly read as a 1) can be identified and the identified codeword matched with a valid codeword when there is a code space of at least 4 between each of the assigned valid code words. In this scenario, if an imaged codeword contains more than one error, the codeword is discarded instead of matched to a valid codeword. In embodiments, the N-bit code has a Hamming weight of at least 4, meaning there are at least four Is in the code which correspond to four distinct read sequences in each of the primary probe pools. In embodiments, each primary nucleic acid probe pool comprises at least 10 different primary nucleic acid probes. In certain embodiments, the primary nucleic acid probes comprise a target sequence, with an average length of between 10 and 200 nucleotides, that hybridize the distinct RNA species.

[0031] Biological Samples

[0032] As used herein, “biological sample” herein refers to a collection of cells obtained from a biological source (e.g., cell culture or tissue from a subject). The tissue may contain nucleated cells with chromosomal material. The source of the tissue sample may be solid tissue, as from a fresh, frozen, fixed, FFPE, and / or preserved organ or tissue sample, or biopsy, or aspirate, or blood or any blood constituents, or bodily fluids, such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid, or cells from any time in gestation or development of the subject. The tissue sample may also be primary or cultured cells or cell lines, or culture tissues. The tissue sample may contain compounds which are not naturally intermixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like. In some embodiments of the invention, the tissue sample is non-hematologic tissue (i.e., not blood or bone marrow tissue). InVIZG019.PCT / USembodiments, the biological sample is fresh frozen or fixed frozen tissue sample. In embodiments, the tissue sample used in the present methods is a formalin-fixed paraffin embedded tissue sample.

[0033] In embodiments, nucleic acid fragmentation can be evaluated and determined using methods well known in the art. For example, to evaluate sample quality for in situ hybridization, it is informative to determine the RNA quality of a tissue block using RNA Integrity Number (RIN) or DV200 values via commercially available instruments such as the BioAnalyzer or TapeStation platforms. Briefly, RNA from the samples are extracted first and measured on either BioAnalyzer or TapeStation. RIN is expressed in values that range from 1-10, where 1 indicates a sample has shorter and more degraded RNA whereas 10 reflects longer and less degraded RNA. Higher RIN scores will have more intact 18S and 28S RNAs. DV200 reflects the percentage of RNA fragments greater than 200 nucleotides in length in tissue samples. Tissues with lower DV200 percentages have shorter and more degraded RNA; conversely, a higher percentage indicates longer, and less degraded RNA molecules present in the tissue.

[0034] In some embodiments, the tissue sample is a tissue section, a clinical smear, or a cultured cell or tissue. In some embodiments, the tissue sample comprises a tissue section. As used herein, “section” of a tissue sample herein refers to a single part or piece of a tissue sample, for example, a thin slice of tissue or cells cut from a tissue sample. It is understood that multiple sections of tissue samples may be taken and subjected to analysis according to the present invention. In some embodiments, the selected portion or section of tissue comprises a homogeneous population of cells. In some embodiments, the selected portion or section of tissue comprises a heterogeneous population of cells. In some embodiments, the selected portion comprises a region of tissue, e.g., the lumen as a non-limiting example. The selected portion can be as small as one cell or two cells, or could represent many thousands of cells, for example.

[0035] Any tissue sample from the subject may be used. Examples of tissue samples that may be used include, but are not limited to, breast, prostate, ovary, colon, lung, endometrium, stomach, salivary gland, or pancreas. The tissue sample can be obtained by a variety of procedures including, but not limited to, surgical excision, aspiration, or biopsy. The tissue may be fresh frozen, fixed frozen or FFPE.

[0036] In some embodiments, the tissue section is a tissue section of brain, adrenal glands, colon, small intestines, stomach, heart, liver, skin, kidney, lung, pancreas, testis, ovary, prostate, uterus,VIZG019.PCT / USthyroid, and spleen of a mammal (e.g., human or mouse). The methods of the present disclosure may be applied to any type of tissue, including, for example, cancer tissue (including from any cancer). In some embodiments, the tissue section is from a solid tumor. In some embodiments, the tissue sample is from mouse small intestine. In some embodiments, the tissue sample is from mouse brain. In some embodiments, the tissue sample is from human liver cancer. In some embodiments, the tissue sample is from human kidney. In some embodiments, the tissue sample is from human lung. In some embodiments, the tissue sample is from human ovarian cancer. In some embodiments, the tissue sample is from human uterus cancer. In some embodiments, the tissue sample is from human lung cancer.

[0037] In some embodiments, the tissue has been stored for a period of time, for example, the period of time that frozen or FFPF, are stored. In some embodiments, the tissue sample is a frozen tissue sample. In some embodiments, the tissue is frozen tissue. In some embodiments, the tissue is paraffin-embedded tissue. In some embodiments, the tissue is formalin-fixed paraffin-embedded tissue. A. Preparation of Tissue SampleObtaining and Fixing Tissue Samples

[0038] Tissue samples can be obtained from an intact organ or tissue using any methods well known to those of skill in the art, e.g., the prior methods used to prepare tissue samples for immunohistochemistry (IHC) or in situ hybridization (ISH) techniques.

[0039] For example, any intact organ or tissue may be cut into reasonably small piece(s) (the size of the cut pieces typically ranges from a few millimeters to a few centimeters) and “fixed” to preserve the positions of the nucleic acids within the sample. Techniques for fixing cells and tissues are known to those of ordinary skill in the art. Non-limiting examples of fixatives include such as formaldehyde, paraformaldehyde, glutaraldehyde, ethanol, methanol, acetone, acetic acid, or the like.

[0040] In some embodiments, the tissue sample is fixed in a solution containing an aldehyde. In some embodiments, the tissue sample is fixed in a solution containing formalin. In some embodiments, the tissue sample is paraffin embedded. In embodiments, the tissue sample is both formalin-fixed and paraffin-embedded (FFPE).

[0041] In addition to intact samples, other samples may be used. In some embodiments, the frozen-sections may be prepared by rehydrating 50 mg of frozen pulverized tissue at room temperature inVIZG019.PCT / USphosphate-buffered saline (PBS) in a small plastic capsule; pelleting the particles by centrifugation; resuspending the particles in a viscous embedding medium (OCT); inverting the capsule and / or pelleting again by centrifugation; snap-freezing in -70 °C isopentane; cutting the plastic capsule and / or removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and / or cutting 25-50 serial sections. Similarly, permanent tissue sections may be prepared involving rehydration of the 50 mg sample in a plastic micro fuge tube; pelleting; resuspending in 10% formalin for a 4 hour fixation; washing / pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue / agar block from the tube; infiltrating and / or embedding the block in paraffin; and / or cutting up to 50 serial permanent sections.

[0042] In some embodiments, the present invention may utilize standard frozen samples, such as those that are embedded in OCT and that are not pulverized, for example, including those used in standard Frozen Section hospital labs.

[0043] Tissue samples are often fixed by conventional methodology. Aldehyde fixatives such as formalin (formaldehyde) and glutaraldehyde are typically used. Tissue samples fixed using other fixation techniques, such as alcohol immersion, are also suitable. See Battifora and Kopmski, J., Histochem. Cytochem., 34:1095 (1986). One of skill in the art will appreciate that the choice of the fixative is determined by the purpose for which the tissue is to be histologically stained or otherwise analyzed. One of skill in the art will also appreciate that the length of fixation depends upon the size of the tissue sample and the fixative used.

[0044] The samples used may also be embedded in paraffin. In some embodiments, the tissue sample is fixed and embedded in paraffin or the like. In some embodiments, the tissue sample is both formalin-fixed and paraffin-embedded. In some embodiments, the formalin-fixed paraffin-embedded (FFPE) tissue block is hematoxylin and eosin (H& E) stained. As commonly known in the art, the tissue sample may be first fixed and is then dehydrated through an ascending series of alcohols, infiltrated and embedded with paraffin or other sectioning media so that the tissue sample may be sectioned. Alternatively, one may section the tissue and fix the sections obtained. Byway of example, the tissue sample may be embedded and processed in paraffin by conventional methodology. Examples of paraffin that may be used include, but are not limited to, Paraplast, Broloid, and Tissuemay. Once the tissue sample is embedded, the sample may be sectioned by a microtome or the like. Once sectioned, the sections may be attached to slides by several standard methods. Examples of slide adhesives include, but are not limited to, silane, gelatin, poly-L-lysine and the like. By way ofVIZG019.PCT / USexample, the paraffin embedded sections may be attached to positively charged slides and / or slides coated with poly-L-lysine.

[0045] In some embodiments, the tissue section may range from about 3 pm to about 100 pm, or any intermediate ranges therewithin. In some embodiments, the tissue section may range from about 10 pm to about 100 pm. In some embodiments, the tissue section may range from about 10 pm to about 50 pm. In some embodiments, the tissue section may range from about 10 pm to about 30 pm. In some embodiments, the tissue section may range from about 10 pm to about 15 pm. In some embodiments, the tissue section may range from about 3 pm to about 15 pm. In some embodiments, the tissue section may range from about 5 pm to about 20 pm. In some embodiments, the tissue section may range from about 15 pm to about 30 pm. In some embodiments, the tissue section may range about 3 pm, about 4 pm, about 5 pm, about 6 pm, about 7 pm, about 8 pm, about 9 pm, about 10 pm, about 11 pm, about 12 pm, about 13 pm, about 14 pm, about 15 pm, or about 20 pm. In some embodiments, the tissue section may range about 30 pm, about 40 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, or about 100 pm.

[0046] Tissue sections can be deparaffinized using methods known in the art and / or commercially available kits. The methods remove the bulk of paraffin from the sample. Various techniques are known for deparaffinizing and include, but are not limited to, washing with an organic solvent or agent to dissolve the paraffin.

[0047] Exemplar deparaffinization solvents include but are not limited to, benzene, toluene, ethylbenzene, xylenes, D-limonene, octane, and mixtures thereof. In certain embodiments, the deparaffinization solvents comprise Ddimonene. These solvents are preferably of high purity, usually greater than 99%. The volume used and the number of washes necessary will depend on the size of the sample and the amount of paraffin to be removed. A sample may be washed between 1 and about 10 times, or between about two and about four times. A typical volume of organic solvent is about 500 ml for a 10 mm tissue sample.

[0048] After deparaffinization, samples may be rehydrated such as by stepwise washing with aqueous lower alcoholic solutions of decreasing concentrations. Ethanol is a preferred lower alcohol for rehydrations while other alcohols may also be used. Non-limiting examples include methanol, isopropanol, and other C1-C5 alcohols. The sample is alternatively vigorously mixed with alcoholicVIZG019.PCT / USsolutions followed by its removal. In some embodiments, deparaffinization and rehydration are carried out simultaneously using a reagent such as EZ-DEWAX™ (BioGenex, San Ramon, CA), for example.

[0049] In some embodiments, the concentration of alcohol is stepwise lowered. In some embodiments, the concentration range of alcohol is decreased stepwise from about 100% to about 70% in water over about three to five incremental steps. In some embodiments, the concentration range of alcohol is decreased stepwise over three incremental steps with 100%, 90%, and 70% respectively.

[0050] In some embodiments of the present disclosure, the samples may be pretreated, such as to facilitate directly or indirectly the methods of the invention. In some embodiments, pretreatment of the tissue increases availability of the target nucleic acid or other targets (e.g., for cell morphology staining). Pretreatments for making targets available (e.g., “antigen retrieval” that retrieves or unmasks the biological markers of interest). An extensive review of antigen retrieval may be found in Shi et al.1997, J Histochem Cytochem, 45(3):327. Antigen retrieval includes a variety of methods by which the availability of the target for interaction with a specific detection reagent is maximized.

[0051] The most common techniques are protease-induced epitope retrieval (PIER) or heat induced epitope retrieval (IIIER). Protease-induced epitope retrieval (PIER) may employ enzymes such as proteinase K, pepsin, trypsin, protease, and any subtypes thereof, in an appropriate buffer to restore the epitope for antibody binding. Heat-induced epitope retrieval (HIER) may employ heat to reverse some cross-links and allow the restoration of epitopes. Citrate buffers, Tris, and EDTA base may be employed as exemplary heat-induced reagents in appropriately pH stabilized manner (e.g., 10 mM sodium citrate, 6.0 pH; 1 mM EDTA, pH 8.0; 10 mM Tris base, 1 mM EDTA solution, 0.05% Tween 20, pH 9.0). Detergents (e.g., Tween 20) may be added to the HIER buffer to increase the epitope retrieval. In certain aspects, many proprietary formulations may be available for the PIER or HIER mediate antigen retrieval.

[0052] Selective staining may be conducted on a tissue section for detection of biological markers and identification of cell types (e.g., nuclear and / or cell morphology stains). To facilitate the specific recognition of biological markers in fixed tissue (e.g., FFPE tissue sample post- deparaffinization and rehydration), it is often necessary to retrieve or unmask the biological markers of interest, through “antigen retrieval” (also called epitope retrieval or antigen unmasking)VIZG019.PCT / US

[0053] In certain embodiments, cellular nucleic acid targets are immobilized in a polymer matrix prior to addition of the bridge probe comprising a moiety that forms a covalent bond with the polymer matrix, 5A Figure 1 “B”.

[0054] It is understood that immobilization of the cellular nucleic acid is a multi-step process, wherein a first and / or second anchoring agents are first added to the tissue sample and a covalent bond is formed between the first anchoring agent and target nucleic acid (as disclosed herein for the first anchor agent of the methods) and the second anchoring agent comprises an oligonucleotide that hybridizes with the target nucleic acid (as disclosed herein for the anchor probe or second anchoring agent of the methods) followed by contact with the polymer matrix wherein both the first and second anchoring agents form covalent bonds with the polymer gel. That entire process immobilizes the target nucleic acid in the polymer gel matrix.

[0055] In embodiments, the target nucleic acid-anchoring agents react to form a covalent bond with the polymer gel before, during or after formation of the polymer matrix.B. Anchoring Agents

[0056] In embodiments, a first and / or second anchoring agents are provided for immobilization of the target nucleic acid (c.g., RNA transcripts) to a polymer matrix, as discussed below. Also, as described below, the first anchoring agent is optionally added with the bridge probe to further anchor the bridge probes in the polymer matrix. In this way, two steps using anchoring agents; first when the cellular nucleic acid is anchored in the polymer matrix, and second to aid in the anchoring of the bridge probes in the polymer matrix.

[0057] In one embodiment, a first anchoring agent is functionalized to comprise a first chemical moiety or reactive group that will form a covalent bond with the target nucleic acid, and a second chemical moiety or reactive group that will form a covalent bond with the polymer gel matrix. In another embodiment, the second anchoring agent comprises an oligonucleotide that hybridizes with the target nucleic acid and a chemical moiety or reactive group that will form a covalent bond with the polymer gel matrix. In certain embodiments, the chemical moiety or reactive group of the second anchoring agent is the same or different as the second chemical moiety of the first anchoring agent. In certain embodiments, the second anchoring agent is also referred to herein as an anchor probe due to the oligonucleotide that hybridizes to the target nucleic acid. In embodiments, the oligonucleotideVIZG019.PCT / USportion of the second anchoring agent comprises a poly-T (thymine residues) for hybridizing with the poly-A tail of an mRNA transcript.

[0058] In some embodiments, the anchor probes may contain sequences complementary to the desired (target) nucleic acid species, e.g., binding to them via base pairing (hybridizing). In embodiments, anchor probes, comprise a chemical moiety or reactive group able to polymerize (e.g., covalent bonding) with a polymer gel matrix.

[0059] In one set of embodiments, the anchoring agents form a covalent bond during the polymerization process with the polymer gel matrix. For example, in the case of polyacrylamide, the anchoring agent may include an acrydite moiety that can polymerize and become incorporated into the polymer. In certain embodiments, the second anchoring agent or anchor probe comprises an oligonucleotide (poly-T) that hybridizes with the poly-A tail of mRNA and an acrydite moiety that forms a covalent bond with polyacrylamide, wherein the gel embedding step utilizes polyacrylamide.

[0060] The anchoring agents may also contain a portion that can interact with and bind to nucleic acid molecules, or other molecules in which immobilization is desired, e.g., proteins or lipids, other desired targets, etc. The immobilization may be covalent or non-covalent. For example, to immobilize a cellular nucleic acid, the anchoring agents may comprise a nucleic acid comprising an acrydite portion (e.g., at the 5’ end, the 3’ end, an internal base, etc.) and a nucleic acid sequence substantially complementary to at least a portion of the target nucleic acid. For instance, the nucleic acid may be complementary to at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides of the nucleic acid. In some cases, the complementarity may be exact (Watson-Crick complementarity), or there may be 1, 2, or more mismatches. In some cases, the anchoring agent can be configured to immobilize mRNA, e.g., in the case of transcriptomic analysis. For instance, in one set of embodiments, the anchoring agent may contain a plurality of thymine nucleotides, e.g., sequentially, for binding to the poly-A tail of an mRNA. Thus, for example, the anchoring agent can have at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more consecutive thymine nucleotides (e.g., a poly-dT portion) within the anchoring agent. In some cases, at least some of the thymine nucleotides may be “locked” thymine nucleotides. These may comprise at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of these thymine nucleotides. In certain embodiments, the locked and non-locked nucleotides may alternate. Such locked thymine nucleotides may be usetul, tor example, to stabilize the hybridization ot the poly-A tails of the mRNA with the anchoring agent.VIZG019.PCT / US

[0061] In embodiments, the methods herein further comprise the use of another anchoring agent, referred to herein as a first anchoring agent, wherein that anchoring agent is functionalized with a first and second chemical moiety for covalent attachment to the target nucleic acid, and to the polymer gel matrix. In certain embodiments, the anchoring agent is a derivatized alkylating agent wherein the alkylating agent has been derivatized with a chemical moiety7or reactive group that will form a covalent bond with the polymer gel matrix. Alkylating agents are well known in the art, and which form a covalent bond with nucleic acid, including RNA, any of which can be derivatized to form a present anchoring agent. In certain embodiments, the anchoring agent is an alkylating agent derivatized with a reactive group that forms a covalent bond with polyacrylamide. In certain embodiments, the anchoring agent is an alkylating agent that comprises an acrydite moiety. In embodiments, alkylating agents are selected from the group consisting of Altretamine, Bendamustine, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine, Ifosfamide, Lomustine, Mechlorethamine, Melphalan, Oxaliplatin, Temozolomide, Thiotepa and Trabectedin. In embodiments, this first anchoring agent is also used with the primary probes to provide multiple anchor sites to the polymer matrix.

[0062] In certain embodiments, the present methods use both an anchoring probe and an anchoring agent (a first and second anchoring agent) for immobilization of the target nucleic acid in the polymer gel matrix. In some embodiments, the nucleic acid targets are immobilized within the gel via both the anchor probes and the anchoring agents bound to the nucleic acid targets.

[0063] In one set of embodiments, nucleic acid molecules may7be immobilized by covalent bonding. For example, in one set of embodiments, an alkylating agent may be used that covalently binds to nucleic acid molecules and contains a second chemical moiety7that can be incorporated into the polyacrylamide as it is polymerized. In yet another set of embodiments, the terminal ribose in an RNA molecule may be oxidized using sodium periodate (or another oxidizing agent) to produce an aldehyde, which may be cross-linked to acrylamide, or other polymer or gel. In other embodiments, chemical agents that are able to modify bases may be used, such as aldehydes, e.g. paraformaldehyde or gluteraldehyde, alkylating agents, or succinimidyl-containing groups; chemical agents that modify7the terminal phosphate, such as carboiimides, e.g., EDC (l-ethyl-3-(3-dimethylaminopropyl) carbodiimide); chemical agents that modify internal sugars, such as p-maleimido-phenyl isocyanate; or chemical agents that modify terminal sugars, such as sodium periodate. In some cases, these chemical agents can carry a second chemical moiety7that can then beVIZG019.PCT / USdirectly cross-linked to the gel or polymer, and / or which can be further modified with a compound that can be directly cross linked to the gel or polymer.

[0064] In yet other embodiments, a nucleic acid may be immobilized using anchor probes having substantially complementary portions to the DNA or RNA. There may be 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50 or more complementary nucleotides between the anchor probe and the nucleic acid.C. Gel polymer matrix

[0065] In embodiments, the methods disclosed herein comprise immobilizing the bridge probes within a polymer matrix. See Figure 1 “A”. In certain embodiments, the method disclosed herein further comprises immobilizing the cellular nucleic acid target within a polymer gel. See Figure 1 “B”.

[0066] The biological sample may be embedded within a matrix that immobilizes nucleic acid targets. For instance, the matrix may comprise a gel or a polymer, such as polyacrylamide. Thus, for example, acrylamide and a suitable cross-linker (e.g., N, N’ -methylenebisacrylamide) can be added to the sample and polymerized to form a gel. The bridge probes or anchoring agents, if present, may include a portion able to polymerize with the gel (e.g., an acrydite moiety) during the polymerization process, and nucleic acids (e.g., mRNAs containing poly-A tails) may then be able to associate with the anchor portion. In such fashion, the mRNAs may be immobilized to the polyacrylamide gel, either directly or via hybridization to the anchored bridge probes. In embodiments, DNA and / or RNA molecules may be immobilized to the polyacrylamide gel using bridge probes having substantially complementary portions to the DNA or RNA. In other embodiments, cellular DNA and / or RNA molecules may be physically tangled within the polyacrylamide gel, e.g., due to their length, to immobilize them to the polyacrylamide gel.

[0067] The sample may be immobilized or embedded within a polymer or a gel, partially or completely. In some cases, the sample may be embedded within a relatively large polymer or gel, which can then be sectioned or sliced in some cases to produce smaller portions for analysis, e.g., using various microtomy techniques commonly available to those of ordinary skill in the art. For instance, tissues or organs may be immobilized within a suitable polymer or gel.

[0068] A variety of polymers may be used in some embodiments. In some cases, the polymer may be selected to be relatively optically transparent. The polymer may also be one that does not significantlyVIZG019.PCT / USdistort during the polymerization process, although in some cases, the polymer may exhibit some distortion. In some cases, the amount of distortion may be determined as a relative change in size that is less than 5, less than 4, less than 3, less than 2, less than 1.5, less than 1.3, or less than 1.2 (i.e., a change in size of 2 means that a sample doubles in linear dimension), or inverses of these (i.e., an inverse change in size of 2 means that a sample halves in linear dimensions).

[0069] Examples of suitable polymers include polyacrylamide and agarose. In embodiments, the polymer is not a hydrogel and / or does not comprise polymers or monomers that swell or expand. A variety of polymers could be used in various embodiments that involve chemical cross links between gel subunits, including but not limited to acrylic acid, acrylamide, ethylene glycol diacrylate, ethylene glycol dimethacrylate, polyethylene glycol dimethacrylate); and / or hydrophobic or hydrogen bonding interactions, such as poly(N-isopropyl acrylamide), methyl cellulose, (ethylene oxide)-(propylene oxide)-(ethylene oxide terpolymers, sodium alginate, poly(vinyl alcohol), alginate, chitosan, gum Arabic, gelatin, and agarose.II. Tissue Clearing

[0070] After immobilization of either the cellular nucleic acids targets to the gel or the bridge probes (if the cellular nucleic acid was not immobilized with anchoring agents), other cellular components (e.g., non-immobilized nucleic acid) within the sample may be removed or degraded.

[0071] By “clearing” a tissue sample or a “cleared” tissue sample, it is meant that the tissue sample is made substantially permeable to light, i.e., transparent, and the optical properties of the sample change to allow more light to pass through the sample. In some embodiments, about 70% or more of the light (e.g., white light, ultraviolet light or infrared light) that is used to illuminate the sample will pass through the sample and illuminate only selected cellular components (e.g., nucleic acids) therein, e.g., 75% or more of the light, 80% or more of the light, 85% or more of the light, 90% or more of the light, 95% or more ot the light, 98% or more of the light, e.g. 100% of the light will pass through the specimen. Any treatment known for tissue clearing may be used to clear the tissue sample in the methods described herein, which are further discussed below.

[0072] Details of tissue clearing have been further discussed in US Patent Publ. No. 2019 / 0264270 published August 29, 2019, entitled “Matrix imprinting and clearing,” the content of which is incorporated herein by reference in its entirety. Such clearance may include removal (e.g., physical removal) of cellular components from the sample, and / or degradation within the sample, such thatVIZG019.PCT / USthey are no longer as prominent within the background. Degradation may include, for example, chemical degradation, enzymatic degradation, or the like.

[0073] In some cases, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the undesired components within the sample may be removed or degraded. Such clearance may include physical removal or degradation of the components (e.g., to smaller components, components that are not fluorescent, etc.). Removal or degradation of such components may decrease background fluorescence or auto fluorescence within the sample during analysis.

[0074] Multiple clearance steps can also be performed in certain embodiments, e.g., to remove or degrade various undesired components.

[0075] For example, enzymes, denaturants, chelating agents, chemical agents, and the like, may break down the proteins into smaller components and / or ammo acids. These smaller components may be easier to remove physically, and / or may be sufficiently small or inert such that they do not significantly affect the background. Similarly, lipids may be removed or degraded from the sample using surfactants or the like. In some cases, one or more of these are used, e.g., simultaneously or sequentially. Nonlimiting examples of suitable enzymes include proteinases such as proteinase K, proteases or peptidases, or digestive enzymes such as trypsin, pepsin, or chymotrypsin. Non-limiting examples of suitable denaturants include guanidine HC1, acetone, acetic acid, urea, or lithium perchlorate. Nonlimiting examples of chemical agents able to denature proteins include solvents such as phenol, chloroform, guanidinium isocyananate, urea, formamide, etc. Non-limiting examples of surfactants include Triton X-100 (polyethylene glycol p-(l,l,3,3-tetramethylbutyl)-phenyl ether), SDS (sodium dodecyl sulfate), Igepal CA-630, or poloxamers. Non-limiting examples of chelating agents include cthylcncdiaminctctraacctic acid (EDTA), citrate, or polyaspartic acid. In some embodiments, compounds such as these may be applied to the sample to remove or degrade proteins, lipids, and / or other components. For instance, a buffer solution (e.g., containing Tris or tris (hydroxymethyl) aminomethane) may be applied to the sample, then removed.

[0076] Non-limiting examples of techniques to remove or degrade RNA include RNA enzymes such as Rnase A, Rnase T, or Rnase H, or chemical agents, e.g., via alkaline hydrolysis (for example, by increasing the pH to greater than 10). Non-limiting examples of systems to remove or degrade sugars or extracellular matrix include enzymes such as chitinase, heparinases, or other glycosylases. Nonlimiting examples of systems to remove or degrade lipids include enzymes such as lipidases, chemicalVIZG019.PCT / USagents such as alcohols (e.g., methanol or ethanol), or detergents such as Triton X-100 or sodium dodecyl sulfate. Many of these are readily available commercially. In this way, the background of the sample may be reduced, which may facilitate analysis of the nucleic acid probes or other desired targets, e.g., using fluorescence microscopy, or other techniques as discussed herein.

[0077] Cellular Nucleic Acid Targets

[0078] The nucleic acid targets may be, for example, DNA, RNA, or other nucleic acids that arc present in a cell within a biological sample.

[0079] In some embodiments, the nucleic acid target is RNA. The RNA may be coding and / or noncoding RNA. Non-limiting examples of RNA that may be studied within the cell include mRNA, siRNA, rRNA, miRNA, tRNA, IncRNA, snoRNAs, snRNAs, exRNAs, piRNAs, or the like.

[0080] The nucleic acids may be endogenous to the cell or added to the cell. For instance, the nucleic acid may be viral, or artificially created. In some cases, the nucleic acid to be determined may be expressed by the cell.

[0081] In some cases, a significant portion of the nucleic acid within the cell may be studied. For instance, in some cases, enough of the RNA present within a cell may be determined so as to produce a partial or complete transcriptome of the cell. In some cases, at least 4 unique mRNA gene transcripts are determined within a cell, and in some cases, at least 3, at least 4, at least 7, at least 8, at least 12, at least 14, at least 15, at least 16, at least 22, at least 30, at least 31, at least 32, at least 50, at least 63, at least 64, at least 72, at least 75, at least 100, at least 127, at least 128, at least 140, at least 255, at least 256, at least 500, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 4,000, at least 5,000, at least 7,500, at least 10,000, at least 12,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 40,000, at least 50,000, at least 75,000, or at least 100,000 types of mRNAs may be determined within a cell.

[0082] In some cases, the transcriptome of a cell may be determined. It should be understood that the transcriptome generally encompasses all RNA transcript molecules produced within a cell, coding and non-coding, not just coding messenger RNA. Thus, for instance, the transcriptome may also include non-coding rRNA, tRNA, siRNA, miRNA, etc. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the transcriptome of a cell may be determined.VIZG019.PCT / US

[0083] The determination of one or more nucleic acids within the sample may be qualitative and / or quantitative. In addition, the determination may also be spatial, e.g., the position of the nucleic acid within the sample may be determined in two or three dimensions. In some embodiments, the positions, number, and / or concentrations of nucleic acids within the cell (or other sample) may be determined.A. Nucleic acid probes

[0084] Bridge Nucleic Acid Probes (“Bridge Probes”)

[0085] Provided herein are bridge nucleic acid probes comprising a moiety that forms a covalent bond with the polymer matrix. See Figure 2. These bridge probes are added to the biological sample, allowed to hybridize to the (anchored) cellular nucleic acid and then immobilized in the polymer matrix. Once complete, cellular components can be cleared trom the sample, the bridge probes are then hybridized with primary nucleic acid probes and imaged using secondary nucleic acid probes (e.g., fluorescent labeled) either simultaneously or sequentially. In alternative embodiments, when the cellular nucleic acid target is immobilized first, the cellular components are cleared before the bridge probes are added. In certain embodiments, an anchoring agent is added to the sample with the bridge nucleic acid probe in situ, wherein the anchoring agent forms a covalent bond with the bridge probe. In embodiments, the anchoring agent is an alkylating agent. In certain embodiments, the alkylating agent is selected from the group consisting of Altretamine, Bendamustine, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine, Ifosfamide, Lomustine, Mechlorethamine, Melphalan, Oxaliplatin, Temozolomide, Thiotepa and Trabectedin. In certain embodiments, the anchoring agent and the bridge probe each comprise an acrydite moiety that covalently binds the polymer matrix. In certain embodiments, the anchoring agent is an alkylating agent derivatized with an acrydite moiety.

[0086] In embodiments, the bridge probe pools (also referred to herein as a “library”) are generated as described in Figure 2 and exemplified in Example 1. The bridge probes of this disclosure comprise a sequence that is complementary to the cellular nucleic acid target(s) and a moiety that forms a covalently bond with the polymer matrix. In embodiments, the bridge probes are generated as a pool, wherein each pool is specific for a distinct nucleic acid target. Once immobilized in the polymer matrix, the bridge probes form a complementary target of the cellular nucleic acid. Accordingly, primary nucleic acid probes are then designed to hybridize to the anchored bridge probe wherein they eachVIZG019.PCT / UScomprise a bridge probe target binding sequence and one or more read sequences. In embodiments, the 5’ end of the bridge probe is modified with an acrydite moiety, or other moieties that can form a covalent bond with the polymer matrix. See Figure 2.

[0087] Primary Nucleic Acid Probes

[0088] Provided herein are primary nucleic acid probes comprising a bridge probe target binding sequence that hybridizes with the anchored bridge probes. See Figure 1. For instance, in one set of embodiments, the primary nucleic acid probes may include smFISH or MERFISH probes, such as those discussed in US Patent No. 11,098,303 or US Patent No. 10,240,146, each incorporated herein by reference in its entirety.

[0089] The primary nucleic acid probes may comprise nucleic acids (or entities that can hybridize to a nucleic acid, e.g., specifically) such as DNA, RNA, LNA (locked nucleic acids), PNA (peptide nucleic acids), or combinations thereof. In some cases, additional components may also be present within the nucleic acid probes, e.g., as discussed below. Any suitable method may be used to introduce nucleic acid probes into a sample.

[0090] The nucleic acid probes are added to the biological sample. Certain aspects of the present invention arc generally directed to nucleic acid probes that arc introduced into a sample. The probes may comprise any of a variety of entities that can hybridize to a nucleic acid, typically by Watson-Crick base pairing, such as DNA, RNA, LNA, PNA, etc., depending on the application. The nucleic acid probe typically contains a target sequence that is able to bind to at least a portion of a target nucleic acid (e.g., bridge probe), in some cases specifically. When introduced into a sample, the nucleic acid probe may be able to bind to a specific target nucleic acid (e.g., an mRNA, or other nucleic acids as discussed herein). In some cases, the nucleic acid probes may be determined using signaling entities (e.g., as discussed below), and / or by using secondary nucleic acid probes able to bind to the nucleic acid probes (i.e., to primary nucleic acid probes). The determination ot such nucleic acid probes is discussed in detail below.

[0091] In some cases, more than one distinct (primary) nucleic acid probe may be applied to a sample, e.g., simultaneously. For example, there may be at least 2, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 300, at least 1,000, at least 3,000, at least 10,000, at least 30,000, at least 50,000, at least 100,000, at least 250,000, at least 500,000, or at least 1,000,000 distinguishable nucleic acid probes that are applied to a sample, e.g., simultaneously or sequentially.VIZG019.PCT / US

[0092] In certain embodiments, the primary oligonucleotide probes comprise a target sequence designed to hybridize with the anchored target nucleic acid. The target sequence may be positioned anywhere within the nucleic acid probe (or primary nucleic acid probe or encoding nucleic acid probe). The target sequence may contain a region that is substantially complementary to a portion of a target nucleic acid. In some cases, the portions may be at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94" o, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary. In some cases, the target sequence may be at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 65, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 nucleotides in length. In some cases, the target sequence may be no more than 500, no more than 450, no more than 400, no more than 350, no more than 300, no more than 250, no more than 200, no more than 175, no more than 150, no more than 125, no more than 100, be no more than 75, no more than 60, no more than 65, no more than 60, no more than 55, no more than 50, no more than 45, no more than 40, no more than 35, no more than 30, no more than 20, or no more than 10 nucleotides in length. Combinations of any of these are also possible, e.g., the target sequence may have a length of between 10 and 30 nucleotides, between 20 and 40 nucleotides, between 5 and 50 nucleotides, between 10 and 200 nucleotides, or between 25 and 35 nucleotides, between 10 and 300 nucleotides, etc. Typically, complementarity is determined on the basis of Watson-Crick nucleotide base pairing.

[0093] The target sequence of a (primary) nucleic acid probe may be determined with reference to a target nucleic acid suspected of being present within a sample. For example, a target nucleic acid to a protein may be determined using the protein’s sequence, by determining die nucleic acids that are expressed to form the protein. In some cases, only a portion of the nucleic acids encoding the protein are used, e.g., having the lengths as discussed above. In addition, in some cases, more than one target sequence that can be used to identify a particular target may be used. For instance, multiple probes can be used, sequentially and / or simultaneously, that can bind to or hybridize to different regions of the same target. Hybridization typically refers to an annealing process by which complementary singlestranded nucleic acids associate tlirough Watson-Crick nucleotide base pairing (e.g., hydrogen bonding, guanine-cytosine and adenine-fhymine) to form double-stranded nucleic acid.

[0094] In some embodiments, a nucleic acid probe, such as a primary nucleic acid probe, may also comprise one or more “read” sequences designed to hybridize with secondary nucleic acid probesVIZG019.PCT / UScomprising a label (e.g., fluorescent label). However, it should be understood that read sequences are not necessary in all cases. In some embodiments, the nucleic acid probe may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more, 20 or more, 32 or more, 40 or more, 50 or more, 64 or more, 75 or more, 100 or more, 128 or more read sequences. The read sequences may be positioned anywhere within the nucleic acid probe. If more than one read sequence is present, the read sequences may be positioned next to each other, and / or interspersed with other sequences. In certain embodiments, the primary oligonucleotide probes comprise one read sequence. In certain other embodiments, the primary oligonucleotide probes comprise two read sequences, which may the same or distinct from each other (e.g., meaning a secondary nucleic acid probe will not hybridize to distinct read sequences).

[0095] The read sequences, if present, may be of any length. If more than one read sequence is used, the read sequences may independently have the same or different lengths. For instance, the read sequence may be at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 65, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 nucleotides in length. In some cases, the read sequence may be no more than 500, no more than 450, no more than 400, no more than 350, no more than 300, no more than 250, no more than 200, no more than 175, no more than 150, no more than 125, no more than 100, be no more than 75, no more than 60, no more than 65, no more than 60, no more than 55, no more than 50, no more than 45, no more than 40, no more than 35, no more than 30, no more than 20, or no more than 10 nucleotides in length. Combinations of any of these are also possible, e.g., the read sequence may have a length of between 10 and 30 nucleotides, between 20 and 40 nucleotides, between 5 and 50 nucleotides, between 10 and 200 nucleotides, or between 25 and 35 nucleotides, between 10 and 300 nucleotides, etc.

[0096] The read sequence may be arbitrary or random in some embodiments. In certain cases, the read sequences arc chosen so as to reduce or minimize homology with other components of the sample, e.g., such that the read sequences do not themselves bind to or hybridize with other nucleic acids suspected of being within the sample. In some cases, the homology may be less than 10%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In some cases, there may be a homology of less than 20 base pairs, less than 18 base pairs, less than 15 base pairs, less than 14 base pairs, less than 13 base pairs, less than 12 base pairs, less than 11 base pairs, or less than 10 base pairs. In some cases, the base pairs are sequential.VIZG019.PCT / US

[0097] In certain embodiments, primary oligonucleotide probes are provided as a pool of probes, wherein each pool of nucleic acid probes hybridize to a distinct target nucleic sequence (e.g., distinct RNA transcript). In embodiments each pool of probes encode, via read sequences, a N-bit binary code that was assigned to each distinct RNA transcript. In certain embodiments, the N-bit binary code has a Hamming weight of at least 2, at least 4, at least 5, at least 6, at least 7 or at least 8, wherein the Hamming weight value is the number of “1” values in the N-bit code and all other positions are “0”. In embodiments the N-bit binary code has a Hamming weight of at least 2, or at least 4, meaning the code contains two or four “1” bit values, respectively, and the other bit positions are “0”. In embodiments, die N-bit binary code has an N value of 3 to 100, with any value thereof possible. In certain embodiments, the binary code is a 4-bit binary code, a 6-bit binary code, a 8-bit binary code, a 16-bit binary code, a 36-bit binary code, a 50-bit binary code, a 54-bit binary code or a 100-bit binary code, or any combination thereof. Each position of the binary code is either a “0” or a “1”, wherein the binding of secondary probes to the read sequence determines if die hybridization read is “0”, wherein no probe binds, or a “1” wherein secondary probe bound to the read sequence of the primary probe. Sequential hybridization and imaging of the secondary read out probes is performed until each position of the N-bit binary code has been read providing a barcode or codeword for the target nucleic acid (e.g. mRNA sequence).

[0098] In one set of embodiments, a population of nucleic acid probes may contain a certain number of read sequences, which may be less than the number of targets of the nucleic acid probes in some cases. Those of ordinary skill in the art will be aware that if there is one signaling entity and n read sequences, then in general 2n-l different nucleic acid targets may be uniquely identified. However, not all possible combinations need be used. For instance, a population of nucleic acid probes may target 12 different nucleic acid sequences, yet contain no more than 8 read sequences. As another example, a population of nucleic acids may target 140 different nucleic acid species, yet contain no more than 16 read sequences. Different nucleic acid sequence targets may be separately identified by using different combinations of read sequences within each probe. For instance, each probe may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. or more read sequences. In some cases, a population of nucleic acid probes may each contain the same number of read sequences, although in other cases, there may be different numbers of read sequences present on the various probes.

[0099] As a non-limiting example, a first nucleic acid probe may contain a first target sequence, a first read sequence, and a second read sequence, while a second, different nucleic acid probe may containVIZG019.PCT / USa second target sequence, the same first read sequence, but a third read sequence instead of the second read sequence. Such probes may thereby be distinguished by determining the various read sequences present or associated with a given probe or location, as discussed herein.

[0100] In addition, the nucleic acid probes (and their corresponding, complementary sites on the encoding probes), in certain embodiments, may be made using only 2 or only 3 of the 4 bases, such as leaving out all the guanosines (Gs) or leaving out all of the cystosines, (Cs) within the probe. Sequences lacking either Gs or Cs may form very little secondary structure in certain embodiments, and can contribute to more uniform, faster hybridization.

[0101] In some embodiments, the nucleic acid probe may contain a signaling entity. It should be understood that signaling entities are not required in all cases, however; for instance, the nucleic acid probe may be determined using secondary nucleic acid probes in some embodiments, as is discussed in additional detail below. Examples of signaling entities that can be used are also discussed in more detail below.

[0102] Other components may also be present within a nucleic acid probe as well. For example, in one set of embodiments, one or more primer sequences may be present, e.g., to allow for enzymatic amplification of probes. Those of ordinary skill in the art will be aware of primer sequences suitable for applications such as amplification (e.g., using PCR or other suitable techniques). Many such primer sequences are available commercially. Other examples of sequences that may be present within a primary nucleic acid probe include, but are not limited to promoter sequences, operons, identification sequences, nonsense sequences, or the like.

[0103] Typically, a primer is a single-stranded or partially double-stranded nucleic acid (e.g., DNA) that serves as a starting point for nucleic acid synthesis, allowing polymerase enzymes such as nucleic acid polymerase to extend the primer and replicate the complementary strand. A primer is (e.g., is designed to be) complementary to and to hybridize to a target nucleic acid. In some embodiments, a primer is a synthetic primer. In some embodiments, a primer is a non-naturally occurring primer. A primer typically has a length of 10 to 50 nucleotides. For example, a primer may have a length of 10 to 40, 10 to 30, 10 to 20, 25 to 50, 15 to 40, 15 to 30, 20 to 50, 20 to 40, or 20 to 30 nucleotides. In some embodiments, a primer has a length of 18 to 24 nucleotides.

[0104] In addition, the components of the nucleic acid probe may be arranged in any suitable order. For instance, in one embodiment, the components may be arranged in a nucleic acid probe as:VIZG019.PCT / USprimer — read sequences — targeting sequence — read sequences — reverse primer. The “read sequences” in this structure may each contain any number (including 0) of read sequences, so long as at least one read sequence is present in the probe. Non-limiting example structures include:• primer — targeting sequence — read sequences — reverse primer,• primer — read sequences — targeting sequence — reverse primer,• targeting sequence — primer — targeting sequence — read sequences — reverse primer, • targeting sequence — primer — read sequences — targeting sequence — reverse primer, • primer — target sequence — read sequences — targeting sequence — reverse primer, • targeting sequence — primer — read sequence — reverse primer,• targeting sequence — read sequence — primer,• read sequence — targeting sequence — primer,• read sequence — primer — targeting sequence — reverse primer, etc.In addition, the reverse primer is optional in some embodiments, including in all of the abovedescribed examples.III. Detection / Imaging of nucleic acid target / probe complex

[0105] After introduction of the primary nucleic acid probes into a sample, the primary probes are hybridized to the anchored bridge probes, which are a copy of the cellular nucleic acid and detection of the primary probes determines the cellular nucleic acid targets.

[0106] In embodiments, the nucleic acid probes may be directly determined by determining signaling entities (if present), and / or the nucleic acid probes may be determined by using one or more secondary nucleic acid probes (also referred to herein as readout probes), in accordance with certain aspects of the invention. As mentioned, in some cases, the determination may be spatial, e.g., in two or three dimensions. In addition, in some cases, the determination may be quantitative, e.g., the amount or concentration of a primary nucleic acid probe (and of a target nucleic acid) may be determined. Additionally, the secondary probes may comprise any of a variety of entities able to hybridize a nucleicVIZG019.PCT / USacid, e.g., DNA, RNA, LNA, and / or PNA, etc., depending on the application. Signaling entities are discussed in more detail below.

[0107] A secondary nucleic acid probe may contain a recognition sequence able to bind to or hybridize with a read sequence of a primary nucleic acid probe. In some cases, the binding is specific, or the binding may be such that a recognition sequence preferentially binds to or hybridizes with only one of the read sequences that are present. The secondary nucleic acid probe may also contain one or more signaling entities. If more than one secondary nucleic acid probe is used, the signaling entities may be the same or different. In embodiments, the secondary nucleic acid probe comprises a fluorescent label and may be referred to herein as a fluorescent secondary nucleic acid probe.

[0108] The recognition sequences may be of any length, and multiple recognition sequences may be of the same or different lengths. If more than one recognition sequence is used, the recognition sequences may independently have the same or different lengths. For instance, the recognition sequence may be at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, or at least 50 nucleotides in length. In some cases, the recognition sequence may be no more than 75, no more than 60, no more than 65, no more than 60, no more than 55, no more than 50, no more than 45, no more than 40, no more than 35, no more than 30, no more than 20, or no more than 10 nucleotides in length. Combinations of any of these are also possible, e.g., the recognition sequence may7have a length of between 10 and 30, between 20 and 40, or between 25 and 35 nucleotides, etc. In one embodiment, the recognition sequence is of the same length as the read sequence. In addition, in some cases, the recognition sequence may be at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% complementary to a read sequence of the primary nucleic acid probe.

[0109] As mentioned, in some cases, the secondary nucleic acid probe may comprise one or more signaling entities. Examples of signaling entities are discussed in more detail below.

[0110] As discussed, in certain aspects of the invention, nucleic acid probes are used that contain various “read sequences.” For example, a population or pool of primary nucleic acid probes may7contain certain “read sequences” which can bind certain of the secondary nucleic acid probes, and the locations of the primary nucleic acid probes are determined within the sample using secondary7nucleic acid probes, e.g., which comprise a signaling entity7. As mentioned, in some cases, a population of readVIZG019.PCT / USsequences may be combined in various combinations to produce different nucleic acid probes, e.g., such that a relatively small number of read sequences may be used to produce a relatively large number of different nucleic acid probes.

[0111] Thus, in some cases, a population (also referred to herein as a “pool”) of primary nucleic acid probes (or other nucleic acid probes) may each contain a certain number of read sequences, some of which are shared between different primary nucleic acid probes such that the total population of primary nucleic acid probes may contain a certain number of read sequences. A population of nucleic acid probes may have any suitable number of read sequences. For example, a population of primary nucleic acid probes may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 etc. read sequences. More than 20 are also possible in some embodiments. In addition, in some cases, a population of nucleic acid probes may, in total, have 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 20 or more, 24 or more, 32 or more, 40 or more, 50 or more, 60 or more, 64 or more, 100 or more, 128 or more, etc. of possible read sequences present, although some or all of the probes may each contain more than one read sequence, as discussed herein. In addition, in some embodiments, the population of nucleic acid probes may have no more than 100, no more than 80, no more than 64, no more than 60, no more than 50, no more than 40, no more than 32, no more than 24, no more than 20, no more than 16, no more than 15, no more than 14, no more than 13, no more than 12, no more than 11, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, or no more than two read sequences present. Combinations of any of these are also possible, e.g., a population of nucleic acid probes may comprise between 10 and 15 read sequences in total.

[0112] As a non-limiting example of an approach to combinatorially producing a relatively large number of nucleic acid probes from a relatively small number of read sequences, in a population of 6 different types of nucleic acid probes, each comprising one or more read sequences, the total number of read sequences within the population may be no greater than 4. It should be understood that although 4 read sequences are used in this example for ease of explanation, in other embodiments, larger numbers of nucleic acid probes may be realized, for example, using 5, 8, 10, 16, 32, etc. or more read sequences, or any other suitable number of read sequences described herein, depending on the application. If each of the primary nucleic acid probes contains two different read sequences, then by using 4 such read sequences (A, B, C, and D), up to 6 probes may be separately identified. It shouldVIZG019.PCT / USbe noted that in this example, the ordering of read sequences on a nucleic acid probe is not essential, i.e., “AB” and “BA” may be treated as being synonymous (although in other embodiments, the ordering of read sequences may be essential and “AB” and “BA” may not necessarily be synonymous). Similarly, if 5 read sequences are used (A, B, C, D, and E) in the population of primary nucleic acid probes, up to 10 probes may be separately identified. For example, one of ordinary skill in the art would understand that, for k read sequences in a population with n read sequences on each probe, up to x different probes may be produced, assuming that the ordering of read sequences is not essential; because not all of the probes need to have the same number of read sequences and not all combinations of read sequences need to be used in every embodiment, either more or less than this number of different probes may also be used in certain embodiments. In addition, it should also be understood that the number of read sequences on each probe need not be identical in some embodiments. For instance, some probes may contain 2 read sequences while other probes may contain 3 read sequences.

[0113] In some aspects, the read sequences and / or the pattern of binding of nucleic acid probes within a sample may be used to define an error-detecting and / or an error-correcting code, for example, to reduce or prevent misidentification or errors of the nucleic acids. Thus, for example, if binding is indicated (e.g., as determined using a signaling entity), then the location may be identified with a “1”; conversely, if no binding is indicated, then the location may be identified with a “0” (or vice versa, in some cases), when using a pool of primary nucleic acid probes comprising read sequences, wherein each pool each pool ot probes encode, via read sequences, a N-bit binary code with a Hamming weight of at least 2 that was assigned to each distinct target nucleic acid (e.g. RNA transcript). Multiple rounds of binding determinations, e.g., using different nucleic acid probes, can then be used to create a “codeword,” e.g., for that spatial location based on binding of the readout probes to the read sequences of the primary probes. In some embodiments, the N-bit binary code may be subjected to error detection and / or correction. For instance, the codewords may be organized such that, if no match is found for a given set of read sequences or binding pattern of nucleic acid probes, then the match may be identified as an error, and optionally, error correction may be applied sequences to determine the correct target for the nucleic acid probes. In some cases, the codewords may have fewer “letters” or positions that the total number of nucleic acids encoded by the codewords, e.g., where each codeword encodes a different nucleic acid.VIZG019.PCT / US

[0114] Such error-detecting and / or the error-correction code may take a variety of forms. A variety of such codes have previously been developed in other contexts such as the telecommunications industry, such as Golay codes or Hamming codes. In one set of embodiments, the read sequences or binding patterns of the nucleic acid probes are assigned such that not every possible combination is assigned.

[0115] For example, if 4 read sequences are possible and a primary nucleic acid probe contains 2 read sequences, then up to 6 primary nucleic acid probes could be identified; but the number of primary nucleic acid probes used may be less than 6. Similarly, for k read sequences in a population with n read sequences on each primary nucleic acid probe, x different probes may be produced, but the number of primary nucleic acid probes that are used may be any number more or less than k. In addition, these may be randomly assigned, or assigned in specific ways to increase the ability to detect and / or correct errors.

[0116] As another example, if multiple rounds of nucleic acid probes are used, the number of rounds may be arbitrarily chosen. It in each round, each target can give two possible outcomes, such as being detected or not being detected, up to 2n different targets may be possible for n rounds of probes, but the number of nucleic acid targets that are actually used may be any number less than 2n. For example, if in each round, each target can give more than two possible outcomes, such as being detected in different color channels, more than 2n (e.g. 3n, 4n...) different targets may be possible for n rounds of probes. In some cases, the number of nucleic acid targets that are actually used may be any number less than this number. In addition, these may be randomly assigned, or assigned in specific ways to increase the ability to detect and / or correct errors.

[0117] For example, in one set of embodiments, the codewords or nucleic acid probes may be assigned within a code space such that the assignments are separated by a Hamming distance, which measures the number of incorrect “reads” in a given pattern that cause the nucleic acid probe to be misinterpreted as a different valid nucleic acid probe. The Hamming weight refers to the distance between the N-bit binary code assigned to a target and thus each pool of primary oligonucleotide probes as encoded via their read sequences. In embodiments a pool of primary probes may have an assigned N-bit binary code with a Hamming weight of at least 4 and a Hamming weight between pools of 4. In that embodiment, errors can both be detected and corrected. In certain cases, the Hamming distance may be at least 2, at least 3, at least 4, at least 5, at least 6, or the like. In addition, in one set of embodiments, the assignments may be formed as a Hamming code, for instance, a Hamming(7, 4)VIZG019.PCT / UScode, a Hamming(15, 11) code, a Hamming(31, 26) code, a Hamming(63, 57) code, a Hamming(127, 120) code, etc. In another set of embodiments, the assignments may form a SECDED code, e.g., a SECDED(8,4) code, a SECDED(16,4) code, a SCEDED(16, 11) code, a SCEDED(22, 16) code, a SEEDED (39, 32) code, a SEEDED (72, 64) code, etc. In yet another set of embodiments, the assignments may form an extended binary Golay code, a perfect binary Golay code, or a ternary Golay code. In another set of embodiments, the assignments may represent a subset of the possible values taken from any of the codes described above.

[0118] For example, a code with the same error correcting properties of the SEEDED code may be formed by using only binary words that contain a fixed number of T bits, such as 4, to encode the targets. In another set of embodiments, the assignments may represent a subset of the possible values taken from codes described above for the purpose of addressing asymmetric readout errors. For example, in some cases, a code in which the number of T’ bits may be fixed for all used binary words may eliminate the biased measurement of words with different numbers of ‘1 ’s when the rate at which ‘0’ bits are measured as ‘l’s or ‘1’ bits are measured as ‘0’s are different.

[0119] Accordingly, in some embodiments, once the codeword is determined (e.g., as discussed herein), the codeword may be compared to the known nucleic acid codewords. If a match is found, then the nucleic acid target can be identified or determined. If no match is found, then an error in the reading of the codeword may be identified. In some cases, error correction can also be applied to determine the correct codeword, and thus resulting in the correct identity of the nucleic acid target. In some cases, the codewords may be selected such that, assuming that there is only one error present, only one possible correct codeword is available, and thus, only one correct identity of the nucleic acid target is possible. In some cases, this may also be generalized to larger codeword spacings or Hamming distances; for instance, the codewords may be selected such that if two, three, or four errors are present (or more in some cases), only one possible correct codeword is available, and thus, only one correct identity of the nucleic acid targets is possible.

[0120] The error-correcting code may be a binary error-correcting code, or it may be based on other numbering systems, e.g., ternary or quaternary error-correcting codes. For instance, in one set of embodiments, more than one type of signaling entity may be used and assigned to different numbers within the error-correcting code. Thus, as a non-limiting example, a first signaling entity (or more than one signaling entity, in some cases) may be assigned as “1” and a second signaling entity (or more than one signaling entity, in some cases) may be assigned as “2” (with “0” indicating no signaling entityVIZG019.PCT / USpresent), and the codewords distributed to define a ternary error-correcting code. Similarly, a third signaling entity may additionally be assigned as “3” to make a quaternary error-correcting code, etc.

[0121] The contents of each of the following references are incorporated herein by reference: US Patent No. 11,098,303, entitled “Systems and Methods for Determining Nucleic Acids”; US Patent No. 10,240,146, entitled “Probe Library Construction”; US Patent Publ. No. 2019-0264270, entitled “Matrix Imprinting and Clearing”; and US Patent Publ. No. 2022-0064697, entitled “Amplification methods and systems for MERFISH and other applications,” for further discussions of Multiplexed Error-Robust Fluorescence In Situ Hybridization (MERFISH) and its examples (e.g., MERFISH probes described herein, signal amplification, determining nucleic acid probes, creating codewords, and error detection and correction, etc.).

[0122] As discussed above, in certain aspects, signaling entities are determined, e.g., to determine nucleic acid probes and / or to create codewords. In some cases, signaling entities within a sample may be determined, e.g., spatially, using a variety of techniques. In some embodiments, the signaling entities may be fluorescent, and techniques for determining fluorescence within a sample, such as fluorescence microscopy or confocal microscopy, may be used to spatially identify the positions of signaling entities within a cell. In some cases, the positions of entities within the sample may be determined in two or even three dimensions. In addition, in some embodiments, more than one signaling entity may be determined at a time (e.g., signaling entities with different colors or emissions), and / or sequentially.

[0123] In addition, in some embodiments, a confidence level for the identified nucleic acid target may be determined. For example, the confidence level may be determined using a ratio of the number of exact matches to the number of matches having one or more one-bit errors. In some cases, only matches having a confidence ratio greater than a certain value may be used. For instance, in certain embodiments, matches may be accepted only if the confidence ratio for the match is greater than about 0.01, greater than about 0.03, greater than about 0.05, greater than about 0.1, greater than about 0.3, greater than about 0.5, greater than about 1, greater than about 3, greater than about 5, greater than about 10, greater than about 30, greater than about 50, greater than about 100, greater than about 300, greater than about 500, greater than about 1000, or any other suitable value. In addition, in some embodiments, matches may be accepted only if the confidence ratio for the identified nucleic acid target is greater than an internal standard or false positive control by about 0.01, about 0.03, about 0.05, about 0.1, about 0.3, about 0.5, about 1, about 3, about 5, about 10, about 30, about 50, about 100, about 300, about 500, about 1000, or any other suitable valueVIZG019.PCT / US

[0124] In some embodiments, the spatial positions of the entities (and thus, nucleic acid probes that the entities may be associated with) may be determined at relatively high resolutions. For instance, the positions may be determined at spatial resolutions of better than about 100 micrometers, better than about 30 micrometers, better than about 10 micrometers, better than about 3 micrometers, better than about 1 micrometer, better than about 800 nm, better than about 600 nm, better than about 500 nm, better than about 400 nm, better than about 300 nm, better than about 200 nm, better than about 100 nm, better than about 90 nm, better than about 80 nm, better than about 70 nm, better than about 60 nm, better than about 50 nm, better than about 40 nm, better than about 30 nm, better than about 20 nm, or better than about 10 nm, etc.

[0125] There are a variety of techniques able to determine or image the spatial positions of entities or targets optically, e.g., using fluorescence microscopy, using radioactivity, via conjugation with suitable chromophores, or the like. For example, various conventional microscopy techniques that may be used in various embodiments of the invention include, but are not limited to, epi-fluorescence microscopy, total-internal-reflectance microscopy, highly inclined thin-illumination (HILO) microscopy, light-sheet microscopy, scanning confocal microscopy, scanning line confocal microscopy, spinning disk confocal microscopy, or other comparable conventional microscopy techniques.

[0126] In some embodiments, in situ hybridization (ISH) techniques for labeling nucleic acids such as DNA or RNA may be used, e.g., where nucleic acid probes may be hybridized to nucleic acids in samples. These may be performed, e.g., at cellular-scale or single-molecule-scale resolutions. In some cases, the ISH probes can be composed of RNA, DNA, PNA, LNA, other synthetic nucleotides, or the like, and / or a combination of any of these. The presence of a hybridized probe can be measured, for example, with radioactivity using radioactively labeled nucleic acid probes, immunohistochemistry using, for example, biotin labeled nucleic acid probes, enzymatic chromophore or fluorophore generation using, for example, probes that can bind enzymes such as horseradish peroxidase and approaches such as tyramide signal amplification, fluorescence imaging using nucleic acid probes directly labeled with fluorophores, or hybridization of secondary nucleic acid probes to these primary probes, with the secondary probes detected via any of the above methods.

[0127] In some cases, the spatial positions may be determined at super resolutions, or at resolutions better than the wavelength of light or the diffraction limit (although in other embodiments, super resolutions are not required). Non-limiting examples include STORM (stochastic opticalVIZG019.PCT / USreconstruction microscopy), STED (stimulated emission depletion microscopy), NSOM (Near-field Scanning Optical Microscopy), 4Pi microscopy, SIM (Structured Illumination Microscopy), SMI (Spatially Modulated Illumination) microscopy, RESOLFT (Reversible Saturable Optically Linear Fluorescence Transition Microscopy), GSD (Ground State Depletion Microscopy), SSIM (Saturated Structured-Illumination Microscopy), SPDM (Spectral Precision Distance Microscopy), Photo-Activated Localization Microscopy (PALM), Fluorescence Photoactivation Localization Microscopy (FPALM), LIMON (3D Light Microscopical Nanosizing Microscopy), Super-resolution optical fluctuation imaging (SOFI), or the like. See, e.g., US Pat. No. 7,838,302, issued November 23, 2010, entitled “Sub-Diffraction Limit Image Resolution and Other Imaging Techniques,” by7Zhuang, et al.; US Pat. No. 8,564,792, issued October 22, 2013, entitled “Sub-diffraction Limit Image Resolution in Three Dimensions,” by7Zhuang, et al.; or WO 2013 / 090360, published June 20, 2013, entitled “High Resolution Dual-Objective Microscopy,” by7Zhuang, et al., each incorporated herein by reference in their entireties.

[0128] In one embodiment, the sample may? be illuminated by? single Gaussian mode laser lines. In some embodiments, the illumination profiled may7be flattened by passing these laser lines through a multimode fiber that is vibrated via piezo-electric or other mechanical means. In some embodiments, the illumination profile may be flattened by? passing single-mode, Gaussian beams through a variety of refractive beam shapers, such as the piShaper or a series of stacked Powell lenses. In yet another set of embodiments, the Gaussian beams may be passed through a variety of different diffusing elements, such as ground glass or engineered diffusers, which may be spun in some cases at high speeds to remove residual laser speckle. In yet another embodiment, laser illumination may7be passed through a series of lenslet arrays to produce overlapping images of the illumination that approximate a flat illumination field.

[0129] In some embodiments, the centroids of the spatial positions of the entities may? be determined. For example, a centroid of a signaling entity? may be determined within an image or series of images using image analysis algorithms known to those of ordinary skill in the art. In some cases, the algorithms may be selected to determine non-overlapping single emitters and / or partially overlapping single emitters in a sample. Non-limiting examples of suitable techniques include a maximum likelihood algorithm, a least squares algorithm, a Bayesian algorithm, a compressed sensing algorithm, or the like. Combinations of these techniques may also be used in some cases.VIZG019.PCT / US

[0130] In addition, the signaling entity may be inactivated in some cases. For example, in some embodiments, a first secondary nucleic acid probe containing a signaling entity" may be applied to a sample that can recognize a first read sequence, then the first secondary nucleic acid probe can be inactivated before a second secondary nucleic acid probe is applied to the sample. If multiple signaling entities are used, the same or different techniques may be used to inactivate the signaling entities, and some or all of the multiple signaling entities may be inactivated, e.g., sequentially or simultaneously.

[0131] Inactivation may be caused by removal of the signaling entity (e.g., from the sample, or from the nucleic acid probe, etc.), and / or by chemically altering the signaling entity in some fashion, e.g., by photobleaching the signaling entity, bleaching or chemically" altering the structure of the signaling entity, e.g., by reduction, etc.). For instance, in one set of embodiments, a fluorescent signaling entity may" be inactivated by chemical or optical techniques such as oxidation, photobleaching, chemically bleaching, stringent washing or enzymatic digestion or reaction by" exposure to an enzyme, dissociating the signaling entity from other components (e.g., a probe), chemical reaction of the signaling entity (e.g., to a reactant able to alter the structure of the signaling entity) or the like. For instance, bleaching may occur by exposure to oxy"gen, reducing agents, or the signaling entity could be chemically cleaved from the nucleic acid probe and washed away via fluid flow.

[0132] In some embodiments, various nucleic acid probes (including primary and / or secondary" nucleic acid probes) may" include one or more signaling entities. If more than one nucleic acid probe is used, the signaling entities may each by the same or different. In certain embodiments, a signaling entity is any entity able to emit light. For instance, in one embodiment, the signaling entity is fluorescent. In other embodiments, the signaling entity may be phosphorescent, radioactive, absorptive, etc. In some cases, the signaling entity is any entity that can be determined within a sample at relatively high resolutions, e.g., at resolutions better than the wavelength of visible light or the diffraction limit. The signaling entity may be, for example, a dye, a small molecule, a peptide or protein, or the like. The signaling entity may be a single molecule in some cases. If multiple secondary" nucleic acid probes are used, the nucleic acid probes may comprise the same or different signaling entities.

[0133] Non-limiting examples of signaling entities include fluorescent entities (fluorophores) or phosphorescent entities, for example, cyanine dyes (e.g., Cy2, Cy3, Cy3B, Cy5, Cy5.5, Cy7, etc.), Alexa Fluor dyes, Atto dyes, photoswtichable dyes, photoactivatable dyes, fluorescent dyes, metal nanoparticles, semiconductor nanoparticles or “quantum dots”, fluorescent proteins such as GFP (Green Fluorescent Protein), or photoactivatable fluorescent proteins, such as PAGFP, PSCFP,VIZG019.PCT / USPSCFP2, Dendra, Dendra2, EosFP, tdEos, mEos2, mEos3, PamCherry, PAtagRFP, mMaple, mMaple2, and mMaple3. Other suitable signaling entities are known to those of ordinary skill in the art. See, e.g., US Pat. No. 7,838,302 or W02015160690A1, each incorporated herein by reference in its entirety. In some cases, spectrally distinct fluorescent dyes may be used.

[0134] In one set of embodiments, the signaling entity may be attached to an oligonucleotide sequence via a bond that can be cleaved to release the signaling entity. In one set of embodiments, a fluorophore may be conjugated to an oligonucleotide via a cleavable bond, such as a photocleavable bond. Non-limiting examples of photocleavable bonds include, but are not limited to, 1-(2-nitrophenyl) ethyl, 2-nitrobenzyl, biotin phosphoramidite, acrylic phosphoramidite, diethylaminocoumarin, 1-(4,5-dimethoxy-2-nitrophenyl)ethyl, cyclo-dodecyl (dimethoxy-2-nitrophenyl) ethyl, 4-aminomethyl-3-nitrobenzyl, (4-nitro-3-(l-chlorocarbonyloxyethyl)phenyl)methyl-S-acetylthioic acid ester, (4-nitro-3-(l-thlorocarbonyloxyethyl)phenyl)methyl-3-(2-pyridyldithiopropionic acid) ester, 3-(4,4’-dimethoxytrityl)-l-(2-nitrophenyl)-propane-l,3-diol-[2- cyanoethyl-(N, N-diisopropyl)]-phosphoramidite, l-[2-nitro-5-(6-trifluoroacetylcaproamidomethyl)phenyl]-ethyl-[2-cyano-ethyl-(N, N-diisopropyl)]-phosphoramidite, l-[2-nitro-5-(6-(4,4’-dimethoxytrityloxy)butyramidomethyl)phenyl] -ethyl- [2-cyanoethyl-(N, N-diisopropyl)]-phosphoramiditc, 1 -[2-nitro-5-(6-(N-(4,4’-dimcth oxytrityl)) -bio tinamidocaproamido- methyl) phenyl] -ethyl-[2-cyanoethyl-(N, N-diisopropyl)]-phosphoramidite, or similar linkers. In another set of embodiments, the fluorophore may be conjugated to an oligonucleotide via a disulfide bond. The disulfide bond may be cleaved by a variety of reducing agents such as, but not limited to, dithiothreitol, dithioerythritol, beta-mercaptoethanol, sodium borohydride, thioredoxin, glutaredoxin, trypsinogen, hydrazine, diisobutylaluminum hydride, oxalic acid, formic acid, ascorbic acid, phosphorous acid, tin chloride, glutathione, thioglycolate, 2,3-dimercaptopropanol, 2-mercap to ethylamine, 2-aminoethanol, tris (2-carboxyethyl) phosphine, bis (2-mercap toethyl) sulfone, N, N’-dimethyl-N, N’-bis (mercaptoacetyl) hydrazine, 3-mercaptopropionate, dimethylformamide, thiopropyl-agarose, tri-n-butylphosphine, cysteine, iron sulfate, sodium sulfite, phosphite, hypophosphite, phosphorothioate, or the like, and / or combinations of any of these. In another embodiment, the fluorophore may be conjugated to an oligonucleotide via one or more phosphorothioate modified nucleotides in which the sulfur modification replaces the bridging and / or non-bridging oxygen. The fluorophore may be cleaved from the oligonucleotide, in certain embodiments, via addition of compounds such as but notVIZG019.PCT / USlimited to iodoethanol, iodine mixed in ethanol, silver nitrate, or mercury chloride. In yet another set of embodiments, the signaling entity may be chemically inactivated through reduction or oxidation. For example, in one embodiment, a chromophore such as Cy5 or Cy7 may be reduced using sodium borohydride to a stable, non-fluorescence state. In still another set of embodiments, a fluorophore may be conjugated to an oligonucleotide via an azo bond, and the azo bond may be cleaved with 2-[(2-N-arylamino)phenylazo]pyridine. In yet another set of embodiments, a fluorophore may be conjugated to an oligonucleotide via a suitable nucleic acid segment that can be cleaved upon suitable exposure to DNAse, e.g., an exodeoxyribonuclease or an endodeoxyribonuclease. Examples include, but are not limited to, deoxyribonuclease I or deoxyribonuclease II. In one set of embodiments, the cleavage may occur via a restriction endonuclease. Non-limiting examples of potentially suitable restriction endonucleases include BamHI, BsrI, Notl, Xmal, PspAI, Dpnl, Mbol, Mnll, Eco57I, Ksp632I, Dralll, Ahall, Smal, Mlul, Ilpal, Apal, Bell, BstEII, TaqI, EcoRI, SacI, Ilindll, Ilaell, Drall, Tsp509I, Sau3AI, PacI, etc. Over 3000 restriction enzymes have been studied in detail, and more than 600 of these are available commercially. In yet another set of embodiments, a fluorophore may be conjugated to biotin, and the oligonucleotide conjugated to avidin or streptavidin. An interaction between biotin and avidin or streptavidin allows the fluorophore to be conjugated to the oligonucleotide, while sufficient exposure to an excess of addition, free biotin could “outcompete” the linkage and thereby cause cleavage to occur. In addition, in another set of embodiments, the probes may be removed using corresponding “toe-hold-probes,” which comprise the same sequence as the probe, as well as an extra number of bases of homology to the encoding probes (e.g., 1-20 extra bases, for example, 5 extra bases). These probes may remove the labeled readout probe through a strand¬ displacement interaction.

[0135] As used herein, the term “light” generally refers to electromagnetic radiation, having any suitable wavelength (or equivalently, frequency). For instance, in some embodiments, the light may include wavelengths in the optical or visual range (for example, having a wavelength of between about 400 nm and about 700 nm, i.e., “visible light”), infrared wavelengths (for example, having a wavelength of between about 300 micrometers and 700 nm), ultraviolet wavelengths (for example, having a wavelength of between about 400 nm and about 10 nm), or the like. In certain cases, as discussed in detail below’, more than one entity may be used, i.e., entities that are chemically different or distinct, for example, structurally. However, in other cases, the entities may be chemically identical or at least substantially chemically identical.VIZG019.PCT / US

[0136] Another aspect of the invention is directed to a computer-implemented method. For instance, a computer and / or an automated system may be provided that is able to automatically and / or repetitively perform any of the methods described herein. As used herein, “automated” devices refer to devices that are able to operate without human direction, i.e., an automated device can perform a function during a period of time after any human has finished taking any action to promote the function, e.g., by entering instructions into a computer to start the process. Typically, automated equipment can perform repetitive functions after this point in time. The processing steps may also be recorded onto a machine-readable medium in some cases.

[0137] For example, in some cases, a computer may be used to control imaging of the sample, e.g., using fluorescence microscopy, STORM or other super-resolution techniques such as those described herein. Tn some cases, the computer may also control operations such as drift correction, physical registration, hybridization and cluster alignment in image analysis, cluster decoding (e.g., fluorescent cluster decoding), error detection or correction (e.g., as discussed herein), noise reduction, identification of foreground features from background features (such as noise or debris in images), or the like. As an example, the computer may be used to control activation and / or excitation of signaling entities within the sample, and / or the acquisition of images of the signaling entities. In one set of embodiments, a sample may be excited using light having various wavelengths and / or intensities, and the sequence of the wavelengths of light used to excite the sample may be correlated, using a computer, to the images acquired of the sample containing the signaling entities. For instance, the computer may apply light having various wavelengths and / or intensities to a sample to yield different average numbers of signaling entities in each region of interest (e.g., one activated entity per location, two activated entities per location, etc.). In some cases, this information may be used to construct an image and / or determine the locations of the signaling entities, in some cases at high resolutions, as noted above.IV. Kits

[0138] The present disclosure provides kits for performing the methods described herein. Kits are provided for preparing biological section samples and determining nucleic acid targets in the sample. In an embodiment, die kit includes bridge probes each modified with a moiety that forms a covalent bond with polymer matrix disclosed herein with other components. In certain embodiments, the kit includes a first and / or second anchoring agent disclosed herein together with one or more other components. In embodiments, kits provided herein further comprise primary nucleic acid probes. InVIZG019.PCT / USan embodiment, the kit also includes labeling agents disclosed herein for identification of cell types or tissue morphology. In some embodiments, the kit includes a number of different labeling agents indicating of a tissue obtained from a particular disease (e.g., solid tumor cancer). In some embodiments, the kit also includes a set of nucleic acid probes (e.g., MERFISH probes described herein) together with one or more other components for MERFISH imaging.

[0139] The one or more other kit components can include one or more buffers; a nuclear counterstain; a whole RNA content counterstain; an imaging buffer; software; and other components. A kit can also include instructions for employing the kit components as well as the use of any other reagent not included in the kit. Instructions can include variations that can be implemented.

[0140] In certain embodiments provided herein are kit components and protocols for preparing biological section samples for transcriptome analysis. In one embodiment, a kit comprises one or more of the following components: deparaffinization buffer, decrosslinking buffer, conditioning buffer, sample prep wash buffer, formamide wash buffer, gel embedding premix, clearing premix, gel coverslip, pre -anchoring activator, anchoring butter and digestion premix. Also provided may be anchor probes for immobilizing target nucleic acid (e.g. RNA transcripts) and target probes and reagents thereof used to specifically detect the target nucleic acid in the prepared sample. In embodiments, kits are provided comprising at least primary probes of this disclosure for anchoring in a polymer matrix.

[0141] In some preferred embodiments, this disclosure provides methods for anchoring within a polymer matrix a complementary target of a distinct nucleic acid species and clearing cellular components, the methods comprising: a) contacting a biological sample comprising the distinct nucleic acid species with a bridge probe and an anchoring agent, wherein the bridge probe comprises an acrydite moiety and hybridizes with the distinct nucleic acid species and the anchoring agent forms a covalent bond with the bridge probe, wherein when hybridized the bridge probe provides a complementary target of the distinct nucleic acid species; b) embedding the biological sample in a polymer matrix wherein the complementary target of the distinct nucleic acid species and the anchoring agent each form a covalent bond with the polymer matrix; and, c) clearing cellular components from the polymer matrix wherein the complementary target of the distinct nucleic acid species remain anchored in the polymer matrix to form a matrix anchored complementary target sample. In some preferred embodiments, the methods can comprise adding RNase to the matrix anchored complementary target sample and clearing the distinct nucleic acid species from the polymerVIZG019.PCT / USmatrix. In some preferred embodiments, the anchoring agent is an alkylating agent, optionally selected from the group consisting of Altretamine, Bendamustine, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine, Ifosfamide, Lomustine, Mechlorethamine, Melphalan, Oxaliplatin, Temozolomide, Thiotepa and Trabectedin. In some preferred embodiments, the anchoring agent and the bridge probe each comprise an acrydite moiety that covalently binds the polymer matrix, the anchoring agent is an alkylating agent derivatized with an acrydite moiety, the distinct nucleic acid species is RNA or DNA, and / or the bridge probe comprises a target sequence that hybridizes to the distinct nucleic acid species and an acrydite moiety that covalently binds the polymer matrix. In some preferred embodiments, the methods can further comprise imaging the anchored complementary target of the distinct nucleic acid species by contacting the anchored complementary target with a primary nucleic acid probe. In some preferred embodiments, the primary nucleic acid probe comprises a target sequence that hybridizes to the complementary target and one or more read sequences. In some preferred embodiments, the primary nucleic acid probes are single molecule (sm)FISH probes or multiplexed error robust fluorescence in situ hybridization (MERFISH) probes. In some preferred embodiments, the methods can further comprise: determining the one or more read sequences based on contacting the primary nucleic acid probe with a plurality of secondary nucleic acid probes comprising a recognition sequence that hybridizes to the read sequence of the primary nucleic acid probe, imaging using multiplexed fluorescence in situ hybridization comprising one or more sequential steps of adding a plurality of secondary nucleic acid probes comprising a label moiety that hybridize to the primary nucleic acid probes, imaging using multiplexed error robust fluorescence in situ hybridization (MERFISH) probes wherein the primary nucleic acid probes are MERFISH probes, contacting the biological sample comprising a plurality of distinct RNA species with a plurality of nucleic acid bridge probe pools each of the bridge probes comprising a target sequence and an acrydite moiety that covalently binds to a polymer matrix, wherein when hybridized the bridge probe pools provide a complementary target of the distinct nucleic acid species in step a), and / or contacting the anchored bridge probes with a plurality of primary nucleic acid probe pools, each of the primary nucleic acid probes comprising a bridge probe target sequence and one or more read sequences, wherein each pool of primary nucleic acid probes hybridize to a distinct complementary target of the distinct RNA species and each pool of primary probes encode a N-bit code that was assigned to each distinct RNA species, wherein each assigned N-bit code is a valid codeword with a Hamming distance of 2 or more between valid codewords. In some preferred embodiments, each of the read sequences correspond to a bit value of 1 for the codewords assignedVIZG019.PCT / USto the distinct RNA species. In some preferred embodiments, the methods can further comprise contacting the primary nucleic acid probes with a plurality of readout probes comprising a fluorescent label, wherein the readout probes hybridize to the read sequences of the primary nucleic acid probes; imaging the readout probes bound to the primary nucleic acid probes; and, repeating steps in one or more sequential hybridization and imaging rounds until all N positions in the N-bit code have been imaged to provide an imaged codeword corresponding to each distinct RNA species in a spatial organization. In some preferred embodiments, the valid codewords can have a Hamming distance equal to or greater than 4 between each of the valid codewords, the N-bit code has a Hamming distance of 4, the imaged codeword is matched to a valid codeword assigned to a distinct RNA species, the Hamming distance is 4 or greater and the imaged codewords are corrected and matched to a valid codeword or discarded, the Hamming distance is 4 or greater and the imaged codewords are matched to a valid codeword or discarded, and / or the RNA species is a RNA transcript. In some preferred embodiments, the methods can comprise determining the spatial organization of the transcriptome from a single cell. In some preferred embodiments, each primary nucleic acid probe pool comprises at least 10 different primary nucleic acid probes, each primary nucleic acid probe pool comprises at least four distinct read sequences, each primary nucleic acid probe pool comprises four distinct read sequences, each primary nucleic acid probe pool comprises at least eight distinct read sequences, the primary nucleic acid probes comprise a target sequence with an average length of between 10 and 200 nucleotides that hybridize the distinct RNA species, and / or each primary nucleic acid probe pool comprises at least 10 different target sequences. In some preferred embodiments, the methods include, after each hybridization, imaging round the fluorescent read out probe is quenched to inactivate it and / or imaging round the fluorescent read out probe is inactivated by chemically or enzymatically cleaving the fluorescent label from the readout probe. In some preferred embodiments, the N-bit code comprises at least a 16-bit code, the plurality of fluorescent readout probes comprise at least two distinct fluorescent labels, the plurality of fluorescent readout probes comprise at least three distinct fluorescent labels, the spatial organization of the distinct RNA species is imaged in 2 dimensions, and / or the spatial organization of the distinct RNA species is imaged in 3 dimensions. In some preferred embodiments, the methods further comprise determining abundance for the distinct RNA species and / or anchoring the distinct nucleic acid species in a polymer matrix prior to step a). In some preferred embodiments, the methods comprise anchoring the distinct nucleic acid species comprises contacting the biological sample with a first and / or second anchoring agent, wherein the first anchoring agent forms a covalent bond with the distinct nucleic acid species and theVIZG019.PCT / USsecond anchoring agent comprises an oligonucleotide that hybridizes with the distinct nucleic acid species. In some preferred embodiments, the second anchoring agent can comprise alternating dT and locked dT portions that hybridizes with the distinct nucleic acid species, the second anchoring agent can comprise a poly-dT portion that hybridizes to the distinct nucleic acid species, the first anchoring agent and the second anchoring agent each comprise an acrydite moiety that covalently binds the polymer matrix, the first anchoring agent is an alkylating agent derivatized with an acrydite moiety, the second anchoring agent comprises a poly-dT portion that hybridizes to the distinct nucleic acid species and an acrydite moiety that covalently binds the polymer matrix, and / or the distinct nucleic acid species is RNA or DNA. In some preferred embodiments, the methods can comprise imaging the complementary target of the distinct nucleic acid species by a method comprising the additional steps of: d) contacting the anchored bridge probes with a plurality of primary nucleic acid probe pools each of the primary nucleic acid probes comprising a bridge probe target sequence and one or more read sequences, wherein each pool of primary nucleic acid probes hybridizes to a distinct complementary target of the distinct RNA species and each pool of primary probes encode a N-bit code that was assigned to each distinct RNA species, wherein each assigned N-bit code is a valid codeword with a Hamming distance of 2 between valid codewords; e) contacting the bound primary nucleic acid probes with a plurality of fluorescent readout probes that hybridize to the read sequences of the primary nucleic acid probes; f) imaging the readout probes bound to the primary nucleic acid probes; and, g) repeating steps e) and f) in one or more sequential hybridization and imaging rounds until all N positions in the N- bit code have been imaged providing an imaged bit code corresponding to each of the distinct nucleic acid species in a spatial organization.

[0142] In some preferred embodiments, dais disclosure provides methods for imaging a complementary target of a distinct nucleic acid species and clearing cellular components, the method comprising: a) contacting a biological sample comprising the distinct nucleic acid species with a bridge probe and an anchoring agent, wherein the bridge probe comprises an acrydite moiety and hybridizes with the distinct nucleic acid species and the anchoring agent forms a covalent bond with the bridge probe, wherein when hybridized with the distinct nucleic acid species the bridge probe provides a complementary target for the distinct nucleic acid species; b) embedding the biological sample in a polymer matrix wherein the complementary target of the distinct nucleic acid species and the anchoring agent each form a covalent bond with the polymer matrix; c) clearing cellular components from the polymer matrix wherein the complementary target of the distinct nucleic acid species remainsVIZG019.PCT / USanchored in the polymer matrix to form a matrix anchored complementary target d) contacting the anchored complementary target with a primary nucleic acid probe comprising a bridge probe hybridization sequence and one or more read sequences; and, e) contacting the primary nucleic acid probe with a plurality of fluorescent readout probes that hybridize to the one or more read sequences of the primary nucleic acid probes and imaging the complementary target of the distinct nucleic acid species. In some embodiments, the methods further comprise adding RNase to the matrix anchored complementary target sample and clearing the distinct nucleic acid species from the polymer matrix. In some embodiments, the anchoring agent is an alkylating agent, optionally selected from the group consisting of Altretamine, Bendamustine, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine, Ifosfamide, Lomustine, Mechlorethamine, Melphalan, Oxaliplatin, Temozolomide, Thiotepa and Trabectedin. In some preferred embodiments, the anchoring agent and the bridge probe each comprise an acrydite moiety that covalently binds the polymer matrix, the anchoring agent is an alkylating agent derivatized with an acrydite moiety, the distinct nucleic acid species is RNA or DNA, the primary nucleic acid probe comprises a target sequence that hybridizes to the bridge probe, one or more read sequences and an acrydite moiety that covalently binds the polymer matrix, and / or the primary nucleic acid probes are single molecule (sm)FISH probes or multiplexed error robust fluorescence in situ hybridization (MERFISH) probes. In some preferred embodiments, the methods further comprise determining read sequences based on contacting the primary nucleic acid probe with a plurality of secondary nucleic acid probes comprising a recognition sequence that hybridizes to the read sequence of the primary nucleic acid probe, repeating step e) is repeated one or more times comprising one or more sequential steps of adding a plurality of secondary nucleic acid probes comprising a label moiety that hybridize to the anchored primary nucleic acid probes, and / or imaging using multiplexed error robust fluorescence in situ hybridization (MERFISH) probes wherein the anchored primary nucleic acid probes are MERFISH probes anchored in the polymer matrix. In some preferred embodiments, the biological sample comprises a plurality of distinct RNA species and step a) comprises contacting the biological sample with a plurality of nucleic acid bridge probe pools and wherein each of the bridge probes comprises a target sequence and an acrydite moiety that covalently binds to a polymer matrix, and when hybridized the bridge probe pools provide a complementary target of the distinct nucleic acid species. In some preferred embodiments, the methods further comprises contacting the anchored bridge probes with a plurality of primary nucleic acid probe pools, each of the primary nucleic acid probes comprising a bridge probe target sequence and one or more read sequences, wherein each pool of primary nucleic acid probesVIZG019.PCT / UShybridizes to a distinct complementary target of the distinct RNA species and each pool of primary nucleic acid probes encodes a different N-bit code assigned to each distinct RNA species, wherein each assigned N-bit code is a valid codeword with a Hamming distance of 2 between valid codewords. In some preferred embodiments, each of the read sequences corresponds to a bit value of 1 for the valid codewords assigned to the distinct RNA species. In some preferred embodiments, the methods further comprise contacting the primary nucleic acid probes with a plurality of readout probes comprising a fluorescent label, wherein the readout probes hybridize to the read sequences of the primary nucleic acid probes; imaging the readout probes bound to the primary nucleic acid probes; and, repeating steps in one or more sequential hybridization and imaging rounds until all N positions in the N-bit code have been imaged to provide an imaged codeword corresponding to each distinct RNA species in a spatial organization. In some preferred embodiments, the valid codewords have a Hamming distance equal to or greater than 4 between each of the valid codewords, the N-bit code has a Hamming weight of 4, each imaged codeword is matched to a valid codeword assigned to a distinct RNA species, the Hamming distance is 4 or greater and the imaged codewords are corrected and matched to a valid codeword or discarded, the Hamming distance is 4 or greater and the imaged codewords are matched to a valid codeword or discarded, and / or the RNA species is a RNA transcript. In some preferred embodiments, the methods further comprise determining the spatial organization of the transcriptome from a single cell. In some preferred embodiments, each primary nucleic acid probe pool comprises at least 10 different primary nucleic acid probes, each primary nucleic acid probe pool comprises at least four distinct read sequences, each primary nucleic acid probe pool comprises four distinct read sequences, each primary nucleic acid probe pool comprises at least eight distinct read sequences, the primary nucleic acid probes comprise a target sequence with an average length of between 10 and 200 nucleotides that hybridize to the distinct RNA species, and / or each primary nucleic acid probe pool comprises at least 10 different target sequences. In some preferred embodiments, the methods further comprise imaging round the fluorescent read out probe is quenched to inactivate after each hybridization. In some preferred embodiments, the methods further comprise imaging round the fluorescent read out probe is inactivated by chemically or enzymatically cleaving the fluorescent label from the readout probe after each hybridization. In some preferred embodiments, the N-bit code comprises at least a 16-bit code, the plurality of fluorescent readout probes comprise at least two distinct fluorescent labels, the plurality of fluorescent readout probes comprise at least three distinct fluorescent labels, the spatial organization of the distinct RNA species is imaged in 2 dimensions, and / or the spatial organization of the distinct RNA species isVIZG019.PCT / USimaged in 3 dimensions. In some preferred embodiments, the methods further comprise determining abundance for the distinct RNA species and / or anchoring the distinct nucleic acid species in a polymer matrix prior to step a). In some preferred embodiments, the methods further comprise anchoring the distinct nucleic acid species by contacting the biological sample with a first and / or second anchoring agent, wherein the first anchoring agent forms a covalent bond with the distinct nucleic acid species and the second anchoring agent comprises an oligonucleotide that hybridizes with the distinct nucleic acid species. In some preferred embodiments, the second anchoring agent comprises alternating dT and locked dT portions that hybridizes with the distinct nucleic acid species, the second anchoring agent comprises a poly-dT portion that hybridizes to the distinct nucleic acid species, the first anchoring agent and the second anchoring agent each comprise an acrydite moiety that covalently binds the polymer matrix, the first anchoring agent is an alkylating agent derivatized with an acrydite moiety, the second anchoring agent comprises a poly-dT portion that hybridizes to the distinct nucleic acid species and an acrydite moiety that covalently binds the polymer matrix, and / or the distinct nucleic acid species is RNA or DNA.

[0143] In some preferred embodiments, this disclosure provides methods for imaging a complementary target of a distinct nucleic acid species and clearing cellular components, the method comprising: a) contacting a biological sample comprising the distinct nucleic acid species with a bridge probe and an anchoring agent, wherein the bridge probe comprises an acrydite moiety and hybridizes with the distinct nucleic acid species and the anchoring agent forms a covalent bond with the bridge probe, wherein when hybridized with the distinct nucleic acid species the bridge probe provides a complementary target for the distinct nucleic acid species; b) embedding the biological sample in a polymer matrix wherein the complementary target of the distinct nucleic acid species and the anchoring agent each form a covalent bond with the polymer matrix; c) clearing cellular components from the polymer matrix wherein the complementary target of the distinct nucleic acid species remains anchored in the polymer matrix to form a matrix anchored complementary target; d) contacting the anchored complementary target with a primary nucleic acid probe comprising a bridge probe hybridization sequence and one or more read sequences; and, e) contacting the primary nucleic acid probe with a plurality of fluorescent readout probes that hybridize to the one or more read sequences of the primary nucleic acid probes and imaging the complementary target of the distinct nucleic acid species. In some embodiments, the methods further comprise adding RNase to the matrix anchored complementary target sample and clearing the distinct nucleic acid species from the polymer matrix.VIZG019.PCT / USIn some embodiments, the anchoring agent is an alkylating agent, optionally wherein the alkylating agent is selected from the group consisting of Altretamine, Bendamustine, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine, Ifosfamide, Lomustine, Mechlorethamine, Melphalan, Oxaliplatin, Temozolomide, Thiotepa and Trabectedin. In some preferred embodiments, the anchoring agent and the bridge probe each comprise an acrydite moiety that covalently binds the polymer matrix, the anchoring agent is an alkylating agent derivatized with an acrydite moiety, the distinct nucleic acid species is RNA or DNA, the primary nucleic acid probe comprises a target sequence that hybridizes to the bridge probe, one or more read sequences and an acrydite moiety that covalently binds the polymer matrix, and / or the primary nucleic acid probes are single molecule (sm)FISH probes or multiplexed error robust fluorescence in situ hybridization (MERFISH) probes. In some preferred embodiments, the methods further comprise determining read sequences based on contacting the primary nucleic acid probe with a plurality of secondary nucleic acid probes comprising a recognition sequence that hybridizes to the read sequence of the primary nucleic acid probe, repeating step e) is repeated one or more times comprising one or more sequential steps of adding a plurality of secondary nucleic acid probes comprising a label moiety that hybridize to the anchored primary nucleic acid probes, and / or imaging using multiplexed error robust fluorescence in situ hybridization (MERFISH) probes wherein the anchored primary nucleic acid probes are MERFISH probes anchored in the polymer matrix. In some preferred embodiments, the biological sample comprises a plurality of distinct RNA species and step a) comprises contacting the biological sample with a plurality of nucleic acid bridge probe pools and wherein each of the bridge probes comprises a target sequence and an acrydite moiety that covalently binds to a polymer matrix, and when hybridized the bridge probe pools provide a complementary target of the distinct nucleic acid species. In some preferred embodiments, the methods further comprises contacting the anchored bridge probes with a plurality of primary nucleic acid probe pools, each of the primary nucleic acid probes comprising a bridge probe target sequence and one or more read sequences, wherein each pool of primary nucleic acid probes hybridizes to a distinct complementary target of the distinct RNA species and each pool of primary nucleic acid probes encodes a different N-bit code assigned to each distinct RNA species, wherein each assigned N-bit code is a valid codeword with a Hamming distance of 2 between valid codewords. In some preferred embodiments, each of the read sequences corresponds to a bit value of 1 for the valid codewords assigned to the distinct RNA species. In some preferred embodiments, the methods further comprise contacting the primary nucleic acid probes with a plurality of readout probes comprising a fluorescent label, wherein the readout probes hybridizeVIZG019.PCT / USto the read sequences of the primary nucleic acid probes; imaging the readout probes bound to the primary nucleic acid probes; and, repeating steps in one or more sequential hybridization and imaging rounds until all N positions in the N-bit code have been imaged to provide an imaged codeword corresponding to each distinct RNA species in a spatial organization. In some preferred embodiments, the valid codewords have a Hamming distance equal to or greater than 4 between each of the valid codewords, the N-bit code has a Hamming weight of 4, each imaged codeword is matched to a valid codeword assigned to a distinct RNA species, the Hamming distance is 4 or greater and the imaged codewords are corrected and matched to a valid codeword or discarded, the Hamming distance is 4 or greater and the imaged codewords are matched to a valid codeword or discarded, and / or the RNA species is a RNA transcript. In some preferred embodiments, the methods further comprise determining the spatial organization of the transcriptome from a single cell. In some preferred embodiments, each primary nucleic acid probe pool comprises at least 10 different primary nucleic acid probes, each primary nucleic acid probe pool comprises at least four distinct read sequences, each primary nucleic acid probe pool comprises four distinct read sequences, each primary nucleic acid probe pool comprises at least eight distinct read sequences, die primary nucleic acid probes comprise a target sequence with an average length of between 10 and 200 nucleotides that hybridize to the distinct RNA species, and / or each primary nucleic acid probe pool comprises at least 10 different target sequences. In some preferred embodiments, the methods further comprise imaging round die fluorescent read out probe is quenched to inactivate after each hybridization. In some preferred embodiments, the methods further comprise imaging round the fluorescent read out probe is inactivated by chemically or enzymatically cleaving the fluorescent label from the readout probe after each hybridization. In some preferred embodiments, the N-bit code comprises at least a 16-bit code, the plurality of fluorescent readout probes comprise at least two distinct fluorescent labels, the plurality of fluorescent readout probes comprise at least three distinct fluorescent labels, the spatial organization of the distinct RNA species is imaged in 2 dimensions, and / or the spatial organization of the distinct RNA species is imaged in 3 dimensions. In some preferred embodiments, the methods further comprise determining abundance for the distinct RNA species and / or anchoring the distinct nucleic acid species in a polymer matrix prior to step a). In some preferred embodiments, the methods further comprise anchoring the distinct nucleic acid species by contacting the biological sample with a first and / or second anchoring agent, wherein the first anchoring agent forms a covalent bond with the distinct nucleic acid species and the second anchoring agent comprises an oligonucleotide that hybridizes with the distinct nucleic acid species. In some preferred embodiments, the secondVIZG019.PCT / USanchoring agent comprises alternating dT and locked dT portions that hybridizes with the distinct nucleic acid species, the second anchoring agent comprises a poly-dT portion that hybridizes to the distinct nucleic acid species, the first anchoring agent and the second anchoring agent each comprise an acrydite moiety that covalently binds the polymer matrix, the first anchoring agent is an alkylating agent derivatized with an acrydite moiety, the second anchoring agent comprises a poly-dT portion that hybridizes to the distinct nucleic acid species and an acrydite moiety that covalently binds the polymer matrix, and / or the distinct nucleic acid species is RNA or DNA.

[0144] In some preferred embodiments, this disclosure provides methods for generating and anchoring within a polymer matrix a complementary target of a plurality of distinct nucleic acid species comprising: a) contacting a sample comprising a plurality of distinct nucleic acid species with a plurality of nucleic acid bridge probe pools comprising bridge probes, each bridge probe comprising a target sequence and a moiety that covalently binds to a polymer matrix, wherein when hybridized the bridge probe pools provide a complementary target of the distinct nucleic acid species; b) polymerizing the polymer matrix within the sample; c) anchoring the complementary target of the distinct nucleic acid species to the polymer matrix wherein the bridge probes covalently bind the polymer matrix; and, d) clearing cellular components from the polymer matrix wherein the complementary target of the distinct nucleic acid species remain anchored in the polymer matrix. In some preferred embodiments, the methods further comprise imaging the complementary target of the distinct nucleic acid species by: e) contacting the bridge probes with a plurality of primary nucleic acid probe pools comprising primary nucleic acid probes, each primary nucleic acid probe comprising a bridge probe target sequence and one or more read sequences, wherein each pool of primary nucleic acid probes hybridize to a distinct complementary target of the distinct nucleic acid species and each pool of probes encode a N-bit code that was assigned to each distinct nucleic acid species; f) contacting the bound hybridized primary nucleic acid probes with a plurality of fluorescent readout probes that hybridize to the read sequences of die primary nucleic acid probes; g) imaging the readout probes bound to die primary nucleic acid probes; and, h) repeating steps f) and g) in one or more sequential hybridization and imaging rounds until all N positions in the N-bit code have been imaged providing an imaged bit code corresponding to each of the distinct nucleic acid species in a spatial organization. In some preferred embodiments, the target sequences bridge two bridge probes when hybridized, the bridge probe is DNA, the plurality of distinct nucleic acid species is RNA, and / or the moiety that covalently binds to a polymer matrix is an acrydite moiety. In some preferred embodiments, the methods furtherVIZG019.PCT / UScomprise adding RNase to the matrix anchored complementary target sample and clearing the distinct nucleic acid species from the polymer matrix. In some preferred embodiments, step a) further comprises contacting the biological sample with an anchoring agent, wherein the bridge probe comprises an acrydite moiety and hybridizes with the distinct nucleic acid species and the anchoring agent forms a covalent bond with the bridge probe. In some preferred embodiments, the anchoring agent is an alkylating agent, optionally selected from the group consisting of Altretamine, Bendamustme, Busulfan, Carboplatm, Carmustine, Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine, Ifosfamide, Lomustine, Mechlorethamine, Melphalan, Oxaliplatin, Temozolomide, Thiotepa and Trabectedin. In some preferred embodiment, the anchoring agent and the bridge probe each comprise an acrydite moiety that covalently binds the polymer matrix, the anchoring agent is an alkylating agent derivatized with an acrydite moiety, each assigned N-bit code is a valid codeword with a Hamming distance of 2 between valid codewords, and / or each of the read sequences correspond to a bit value of 1 for the codewords assigned to the distinct RNA species. In some preferred embodiments, the methods further comprise contacting the primary nucleic acid probes with a plurality of readout probes comprising a fluorescent label, wherein the readout probes hybridize to the read sequences of the primary nucleic acid probes; imaging the readout probes bound to the primary nucleic acid probes; and, repeating steps in one or more sequential hybridization and imaging rounds until all N positions in the N-bit code have been imaged providing an imaged codeword corresponding to each distinct RNA species in a spatial organization. In some preferred embodiments, the valid codewords have a Hamming distance equal to or greater than 4 between each of the valid codewords, the N-bit code has a Hamming weight of 4, the imaged codeword is matched to a valid codeword assigned to a distinct RNA species, the Hamming distance is 4 or greater and the imaged codewords are corrected and matched to a valid codeword or discarded, the Hamming distance is 4 or greater and the imaged codewords are matched to a valid codeword or discarded, and / or the RNA species is a RNA transcript. In some preferred embodiments, the methods further comprise determining the spatial organization of the transcriptome from a single cell. In some preferred embodiments, each primary nucleic acid probe pool comprises at least 10 different primary nucleic acid probes, each primary nucleic acid probe pool comprises at least four distinct read sequences, each primary nucleic acid probe pool comprises four distinct read sequences, each primary nucleic acid probe pool comprises at least eight distinct read sequences, the primary nucleic acid probes comprise a target sequence, with an average length of between 10 and 200 nucleotides, that hybridize the distinct RNA species, and / or each primary nucleic acid probe pool comprises at leastVIZG019.PCT / US10 different target sequences. In some preferred embodiments, the methods further comprise imaging round the fluorescent readout probe is quenched to inactivate after each hybridization. In some preferred embodiments, the methods further comprise, after each hybridization and imaging round, inactivating the fluorescent readout probe by chemically or enzymatically cleaving the fluorescent label from the readout probe. In some preferred embodiments, the N-bit code comprises at least a 16-bit code, the plurality of fluorescent readout probes comprise at least two distinct fluorescent labels, the plurality of fluorescent readout probes comprise at least three distinct fluorescent labels, the spatial organization of the distinct RNA species is imaged in 2 dimensions, and / or the spatial organization of the distinct RNA species is imaged in 3 dimensions. In some preferred embodiments, the methods further comprise determining abundance for the distinct RNA species, anchoring the distinct nucleic acid species in a polymer matrix prior to step a), optionally wherein anchoring the distinct nucleic acid species comprises contacting the biological sample with a first and / or second anchoring agent, wherein the first anchoring agent forms a covalent bond with the distinct nucleic acid species and the second anchoring agent comprises an oligonucleotide that hybridizes with the distinct nucleic acid species. In some preferred embodiments, the second anchoring agent comprises alternating dT and locked dT portions that hybridizes with the distinct nucleic acid species, the second anchoring agent comprises a poly-dT portion that hybridizes to the distinct nucleic acid species, the first anchoring agent and the second anchoring agent each comprise an acrydite moiety that covalently binds the polymer matrix, the first anchoring agent is an alkylating agent derivatized with an acrydite moiety, the second anchoring agent comprises a poly-dT portion that hybridizes to the distinct nucleic acid species and an acrydite moiety that covalently binds the polymer matrix, and / or the distinct nucleic acid species is RNA or DNA.

[0145] In some preferred embodiments, the this disclosure provides methods for anchoring within a polymer matrix a complementary target of a distinct nucleic acid species and clearing cellular components comprising: a) contacting a biological sample with a first and / or second anchoring agent, wherein the first anchoring agent forms a covalent bond with the distinct nucleic acid species and the second anchoring agent comprises an oligonucleotide that hybridizes with the distinct nucleic acid species and wherein the first anchoring agent and the second anchoring agent each comprise an acrydite moiety that covalently binds to the polymer matrix; b) embedding the biological sample in a polymer matrix wherein the first and second anchoring agents each form a covalent bond with the polymer matrix; c) clearing the cellular components from the polymer matrix wherein the distinctVIZG019.PCT / USnucleic acid species remains anchored in the polymer matrix; d) contacting the anchored distinct nucleic acid species with a bridge probe and the first anchoring agent, wherein the bridge probe comprises an acrydite moiety and hybridizes with the distinct nucleic acid species and the first anchoring agent forms a covalent bond with the bridge probe, wherein when hybridized the bridge probe provides a complementary target of the distinct nucleic acid species; and, e) embedding the bridge probe sample in a polymer matrix wherein the complementary target of the distinct nucleic acid species and the anchoring agent each form a covalent bond with the polymer matrix to form a matrix anchored complementary target sample. In some preferred embodiments, the methods further comprises adding RNase to the matrix anchored complementary target sample and clearing die distinct nucleic acid species from the polymer matrix. In some preferred embodiments, the first anchoring agent is an alkylating agent, optionally wherein the alkylating agent is selected from the group consisting of Altretamine, Bendamustine, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine, Ifosfamide, Lomustine, Mechlorethamine, Melphalan, Oxaliplatin, Temozolomide, Thiotepa and Trabectedin. In some preferred embodiments, the first anchoring agent and die bridge probe each comprise an acrydite moiety that covalently binds the polymer matrix, the first anchoring agent is an alkylating agent derivatized with an acrydite moiety, and / or the distinct nucleic acid species is RNA or DNA. In some preferred embodiments, the bridge probe comprises a target sequence that hybridizes to the distinct nucleic acid species and an acrydite moiety that covalentiy binds the polymer matrix. In some preferred embodiments, die methods further comprise imaging the anchored complementary target of the distinct nucleic acid species by contacting the anchored complementary target with a primary nucleic acid probe. In some preferred embodiments, the primary nucleic acid probe comprises a target sequence that hybridizes to the complementary target and one or more read sequence and / or the primary nucleic acid probes are single molecule (sm)FISH probes or multiplexed error robust fluorescence in situ hybridization (MERFISH) probes. In some preferred embodiments, the methods further comprise determining die one or more read sequences based on contacting the primary nucleic acid probe with a plurality of secondary nucleic acid probes comprising a recognition sequence that hybridizes to die read sequence of the primary nucleic acid probe, imaging using multiplexed fluorescence in situ hybridization comprising one or more sequential steps of adding a plurality of secondary nucleic acid probes comprising a label moiety that hybridize to the primary nucleic acid probes, and / or imaging using multiplexed error robust fluorescence in situ hybridization (MERFISH) probes wherein die primary nucleic acid probes are MERFISH probes. In some preferred embodiments, the anchored distinct nucleic acid speciesVIZG019.PCT / UScomprise distinct RNA species and step d) comprises contacting the anchored distinct RNA species with a plurality of nucleic acid bridge probe pools each of the bridge probes comprising a target sequence and an acrydite moiety that covalently binds to a polymer matrix, wherein when hybridized the bridge probe pools provide a complementary target of the distinct RNA species. In some preferred embodiments, the methods further comprise contacting the anchored bridge probes with a plurality of primary nucleic acid probe pools, each of the primary nucleic acid probes comprising a bridge probe target sequence and one or more read sequences, wherein each pool of primary nucleic acid probes hybridize to a distinct complementary target of the distinct RNA species and each pool of primary probes encode a different N-bit code that was assigned to each distinct RNA species, wherein each assigned N-bit code is a valid codeword with a Hamming distance of 2 between valid codewords. In some preferred embodiments, each of the read sequences correspond to a bit value of 1 for the codewords assigned to die distinct RNA species. In some preferred embodiments, the metiiods furtiier comprise contacting the primary nucleic acid probes with a plurality of readout probes comprising a fluorescent label, wherein the readout probes hybridize to the read sequences of the primary nucleic acid probes; imaging the readout probes bound to the primary nucleic acid probes; and, repeating steps in one or more sequential hybridization and imaging rounds until all N positions in the N-bit code have been imaged providing an imaged codeword corresponding to each distinct RNA species in a spatial organization. In some preferred embodiments, the valid codewords have a Hamming distance equal to or greater than 4 between each of the valid codewords, the N-bit code has a Hamming weight of 4, the imaged codeword is matched to a valid codeword assigned to a distinct RNA species, the Hamming distance is 4 or greater and the imaged codewords are corrected and matched to a valid codeword or discarded, the Hamming distance is 4 or greater and the imaged codewords are matched to a valid codeword or discarded, and / or each distinct RNA species is a RNA transcript. In some preferred embodiments, the methods further comprise determining the spatial organization of the transcriptome from a single cell. In some preferred embodiments, each primary nucleic acid probe pool comprises at least 10 different primary nucleic acid probes, each primary nucleic acid probe pool comprises at least four distinct read sequences, each primary nucleic acid probe pool comprises four distinct read sequences, each primary nucleic acid probe pool comprises at least eight distinct read sequences, the primary nucleic acid probes comprise a target sequence, with an average length of between 10 and 200 nucleotides, that hybridize the distinct RNA species, and / or each primary nucleic acid probe pool comprises at least 10 different target sequences. In some preferred embodiments, the methods further comprise, after each hybridization and imaging round,VIZG019.PCT / USquenching the fluorescent read out probe to inactivate it. In some preferred embodiments, after each hybridization and imaging round, the fluorescent read out probe is inactivated by chemically or enzymatically cleaving the fluorescent label from die readout probe. In some preferred embodiments, the N-bit code comprises at least a 16-bit code, the plurality of fluorescent readout probes comprise at least two distinct fluorescent labels, the plurality of fluorescent readout probes comprise at least three distinct fluorescent labels, the spatial organization of the distinct RNA species is imaged in 2 dimensions, and / or the spatial organization of the distinct RNA species is imaged in 3 dimensions. In some preferred embodiments, the methods further comprise determining abundance for the distinct RNA species. In some preferred embodiments, die second anchoring agent comprises alternating dT and locked dT portions that hybridizes with the distinct nucleic acid species, the second anchoring agent comprises a poly-dT portion that hybridizes to the distinct nucleic acid species, the first anchoring agent and the second anchoring agent each comprise an acrydite moiety that covalently binds the polymer matrix, the second anchoring agent comprises a poly-dT portion that hybridizes to the distinct nucleic acid species and an acrydite moiety that covalently binds the polymer matrix.

[0146] In some preferred embodiments, the biological sample can be deparaffinized and rehydrated prior to step (a), the polymer matrix can comprise polyacrylamide. In some preferred embodiments, the cellular components comprise proteins, lipids, DNA, RNA, or extracellular matrix. In some preferred embodiments, clearing the cellular components comprises removing or degrading proteins from the biological sample. In some preferred embodiments, clearing the cellular components comprises removing or degrading lipids from the biological sample. In some preferred embodiments, clearing the cellular components comprises removing or degrading target RNA from the biological sample. In some preferred embodiments, clearing die cellular components comprises removing or degrading extracellular matrix from the biological sample. In some preferred embodiments, clearing the cellular components comprises exposing the biological sample to an enzyme able to degrade a protein. In some preferred embodiments, clearing the cellular components comprises exposing the biological sample to an enzyme able to degrade DNA. In some preferred embodiments, clearing the cellular components comprises exposing the biological sample to an enzyme able to degrade RNA. In some preferred embodiments, clearing die cellular components comprises exposing the biological sample to an enzyme able to degrade sugars or sugar-modified biomolecules. In some preferred embodiments, clearing the cellular components comprises exposing the biological sample to a detergent. In some preferred embodiments, clearing the cellular components comprises exposing theVIZG019.PCT / USgel to a proteinase. In some preferred embodiments, clearing the cellular components comprises exposing the polymer matrix to guanidine HCl In some preferred embodiments, clearing the cellular components comprises exposing the gel to Triton X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl) -phenyl ether). In some preferred embodiments, clearing the cellular components comprises exposing the polymer matrix to sodium dodecyl sulfate. In some preferred embodiments, clearing the cellular components comprises exposing the polymer matrix to ethylenediaminetetraacetic acid.

[0147] In some preferred embodiments, this disclosure provides methods in which the imaging comprises using epi-fluorescence microscopy, total-internal-reflectance microscopy, highly inclined thm-illumination (HILO) microscopy, light-sheet microscopy, scanning confocal microscopy, scanning line confocal microscopy, spinning disk confocal microscopy, or other comparable conventional microscopy techniques.

[0148] In some preferred embodiments, this disclosure provides bridge nucleic acid probes comprising: an acrydite moiety and a target sequence configured to hybridize with a distinct nucleic acid species in a biological sample. In some preferred embodiments, this disclosure provides a plurality of bridge nucleic acid probe pools comprising bridge probes, the bridge probes comprising: a target sequence and an acrydite moiety, wherein each bridge nucleic acid probe pool is configured to hybridize to a distinct RNA species in a sample. In some preferred embodiments, the bridge probes comprise a target sequence, with an average length of between 10 and 200 nucleotides, configured to hybridize the distinct RNA species. In some preferred embodiments, each bridge nucleic acid probe pool comprises at least 10 different target sequences. In some preferred embodiments, the bridge nucleic acid probes are covalently bonded to an anchoring agent. In some preferred embodiments, the anchoring agent is an alkylating agent, optionally selected from the group consisting of Altretamine, Bendamustine, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine, Ifosfamide, Lomustine, Mechlorethamine, Melphalan, Oxaliplatin, Temozolomide, Thiotepa and Trabectedin. In some preferred embodiments, the anchoring agent and the bridge nucleic acid probes each comprise an acrydite moiety configured to covalently bind a polymer matrix, the anchoring agent is an alkylating agent derivatized with an acrydite moiety, the bridge nucleic acid probes are embedded in a polymer matrix and the anchoring agent and acrydite moiety of the bridge nucleic acid probe are each covalently bonded the polymer matrix, the polymer matrix is a polyacrylamide gel, the bridge nucleic acid probe pools are hybridized to a plurality ofVIZG019.PCT / USprimary nucleic acid probe pools each of the primary nucleic acid probes comprising a bridge probe target sequence and one or more read sequences, wherein each pool of primary nucleic acid probes hybridize to a distinct bridge nucleic acid probe pool and each pool of primary probes encode a N-bit code that was assigned to each distinct RNA species, each assigned N-bit code is a valid codeword, the valid codewords have a Hamming distance of 2 between valid codewords, each of the read sequences in a primary probe pool correspond to a bit value of 1 for the codewords assigned to the distinct RNA species, the valid codewords have a Hamming distance equal to or greater than 4 between each of the valid codewords, each primary nucleic acid probe pool comprises at least 10 different primary nucleic acid probes, each primary nucleic acid probe pool comprises at least four distinct read sequences, each primary nucleic acid probe pool comprises four distinct read sequences, each primary nucleic acid probe pool comprises at least eight distinct read sequences, the primary nucleic acid probes comprise a target sequence, with an average length of between 10 and 200 nucleotides, configured to hybridize the distinct bridge nucleic acid probe pools, each primary nucleic acid probe pool comprises at least 10 different target sequences, and / or the N-bit code comprises at least a 16-bit code.

[0149] Although embodiments of the invention are explained in detail, it is to be understood that other embodiments are contemplated. Accordingly, it is not intended that the invention is limited in its scope to the details of construction and arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or carried out in various ways. Also, in describing the embodiments, specific terminology will be resorted to for the sake of clarity.

[0150] It must also be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, reference to “a probe” is intended to also include a plurality of probes and reference to “a target” is intended to also include a plurality of targets and the like.

[0151] The term “or” is used herein in the inclusive sense, i.e., equivalent to “and / or” unless the context clearly requires otherwise.

[0152] Numeric ranges are inclusive of the numbers defining the range. Measured and measurable values are understood to be approximate, taking into account significant digits and the error associated with the measurement. Accordingly, unless indicated to the contrary, the numerical parameters setVIZG019.PCT / USforth in the following description and appended claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0153] The term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, or a degree of variation that does not substantially affect the properties of the described subject matter.

[0154] The use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” is not intended to be limiting, and means that at least the named compound, element, particle, or method step is present in the composition or article or method, but does not exclude the presence of other compounds, materials, particles, method steps, even if the other such compounds, material, particles, method steps have the same tunction as what is named.

[0155] Also, in describing the embodiments, terminology will be resorted to for the sake of clarity. It is intended that each term contemplates its broadest meaning as understood by those skilled in the art and includes all technical equivalents which operate in a similar manner to accomplish a similar purpose.

[0156] It is also to be understood that the mention of one or more method steps does not preclude the presence of additional method steps or intervening method steps between those steps expressly identified. Similarly, it is also to be understood that the mention of one or more components in a fabric or system does not preclude the presence of additional components or intervening components between those components expressly identified.

[0157] The skilled artisan will understand that the figures, described above, and example, described below, are for illustration purposes only. Neither the figures nor the examples are intended to limit the scope of the disclosed teachings in anyway.VIZG019.PCT / USV. EXAMPLES

[0158] The following examples are provided to illustrate certain disclosed embodiments and are not to be construed as limiting the scope of this disclosure in any way.

[0159] Example 1. Generation of Bridge Nucleic Acid Probe Pools with an Acrydite Moiety

[0160] This example illustrates generation of a bridge nucleic acid probes (“bridge probes”) and pools thereof to be used with the methods of this disclosure. In embodiments, each bridge probe comprises an acrydite moiety (for covalent attachment to a polymer matrix) and a target hybridization sequence (for binding to a nucleic acid target (also referred to herein as “distinct nucleic acid species”) in a biological sample). The bridge probes are grouped into pools based on the target hybridization sequence wherein each pool is specific for a distinct nucleic acid target providing a complementary target of the distinct nucleic acid species. This example used a 5’ acrydite-modified oligo pool (“bridge probe pool”) and illustrated the use of bridge probes in RNase-insensitive methods and applications for multiplexed imaging of nucleic acid analytes, according to certain embodiments of the invention. See Figure 2.

[0161] Tn embodiments, an array-synthesized complex oligo pool, containing approximately thousands to hundreds of thousand sequences, was used as a template for the enzymatic amplification of the primary nucleic acid probe. Each template sequence in the oligo pool contained a central target region (e.g., bridge probe target hybridization sequence), a forward primer sequence and a T7 promoter sequence. In foe first step, foe template was amplified through RT-PCR on BioRad CFX96 qPCR machine by mixing the following reagents: 20X EvaGreen® Dye (Biotium 31000), forward primer (IDT, 100 uM), reverse primer (IDT, lOOuM), 100 ng template, nuclease free water and 2X Phusion Hot Start II DNA Polymerase (ThermoFisher F549L). The thermocycling program used for RT-PCR was the following: a) 98°C for 3 minutes; b) 98°C for 10 seconds; c) 50°C for 10 seconds; d) 72°C for 15 seconds; e) measure fluorescence; f) 72°C for 10 seconds; g) Repeat steps b through f until the signal amplification rate decreases. The amplified products were then purified through Spin V columns (Zymo, CIO 12-50) and diluted in water. The RT-PCR product was in vitro transcribed into RNA using a Quick HiScribe T7 Polymerase kit (NEB E2040S) for 12-18 hours, column purified, and the RNA product was resuspended in RNase-free water. The RNA product was reverse-transcribed into DNA using Maxima H Minus Reverse Transcriptase (ThermoFisher, EP0751) for 1.5 hours at 50°C. The product was treated with pH 90.25M NaOH, 0.125M EDTA at 95°C for 20 minutes, thenVIZG019.PCT / USneutralized with IM HCL to pH 7, and column purified. In this step, the RNA was reverse transcribed back into DNA via a 5’acrydite-modified forward primer, allowing acrydite modification to be added to each ssDNA. Acrydite is an attachment chemistry based on an acrylic phosphoramidite that can be added to oligonucleotides as a 5’-modification. Acrydite-modified oligonucleotides covalently react with thiol-modified surfaces or can be incorporated into polyacrylamide gels during polymerization. After purification, the product (“bridge nucleic acid probe comprising an acrydite moiety”) was then concentrated using a standard ethanol precipitation technique, and the single-stranded DNA pellet was then resuspended in RNase-free water.

[0162] Example 2: In situ single-cell transcriptomic imaging using anchored bridge probes and MERFISH primary probes in biological samples (“Protocol A”) or in a sample with fragmented Nucleic Acid (NA) or FFPE samples (“Protocol B”)

[0163] This example illustrates a sample protocol for anchoring bridge probes comprising an acrydite moiety in a polymer matrix (“Protocol A”) or prior to anchoring the cellular nucleic acid (NA, e.g., RNA) in a polymer matrix and then adding the bridge probes (“Protocol B”). The latter is useful for FFPE samples, or any sample wherein the nucleic acid is partially degraded. One of skill in the art would understand which protocol to use based on their sample properties. It is understood that Protocol A or Protocol B can be used with any downstream in situ imaging, not just MERFISH imaging, and for anchoring fragmented RNA and that this example is provided for illustrative purposes only and not to imply any limitation on the methods described herein. Protocol A and Protocol B are illustrated in Fig. 1. Comparative Protocol C is a standard AIERFISH methods.

[0164] In embodiments, cell or tissue samples are fixed and permeabilized. Bridge probes, prepared according to Example 1, arc added to the sample preparation, allowed to incubate and hybridize with the cellular RNA. To further aid immobilization of the bridge probes, an anchoring agent is added to the biological sample which forms a covalent bond with the nucleic acid. Next the biological sample is embedded in a hydrogel (e.g. polyacrylamide gel) and the acydrite moieties of the bridge probe and anchoring agent are polymerized with the polymer matrix forming covalent bonds; the cellular components are removed (e.g. cleared) chemically and / or enzymatically, leaving a non-cellular matrix to which the bridge probes, and the hybridized cellular RNA, are chemically crosslinked (e.g., anchored). An RNase treatment may be added which removes the RNA target leaving the bridge probe anchored in the polymer matrix as a proxy for the cellular RNA target. Primary probes (e.g.,VIZG019.PCT / USencoding probes) are added which bind to the target sequence of the bridge probes, followed by labeled secondary probes and imaging.

[0165] In certain other embodiments, especially when the sample is suspected to contain fragmented nucleic acid (e.g., FFPE samples) the cellular nucleic acid is first anchored in a polymer matrix and then the bridge probes are added and also anchored in another layer of polymer matrix. The cellular nucleic acid is anchored using a first and / or second anchoring agent, wherein the first anchoring agent forms a covalent bond with the cellular nucleic acid and the second anchoring agent comprises an oligonucleotide that hybridizes with the cellular nucleic acid and wherein the first anchoring agent and the second anchoring agent each comprise an acrydite moiety that covalently binds to the polymer matrix. Next the biological sample is embedded in a hydrogel (e.g. polyacrylamide gel) and the acydrite moieties of the first and / or second anchoring agent are polymerized with the polymer matrix forming covalent bonds; the cellular components are removed (e.g. cleared) chemically and / or enzymatically, leaving polymer matrix crosslinked to the cellular nucleic acid (e.g., RNA), to which the bridge probes, and the hybridized cellular RNA, are chemically crosslinked (e.g., anchored). Bridge probes, prepared according to Example 1, are added to the anchored nucleic acid, allowed to incubate and hybridize with the cellular RNA. The first anchoring agent is then added forming a covalent bond with the bridge probes and upon embedding with the polymer matrix is cross linked with the matrix. An RNase treatment may be added which removes the RNA target leaving the bridge probe anchored in the polymer matrix as a proxy for the cellular RNA target. Primary probes (e.g., encoding probes) are added which bind to the target sequence of the bridge probes, followed by labeled secondary probes and imaging. See Figure 1.

[0166] Example 3: Multiplexed imaging of 130 RNA species with bridge-mediated encoding probe hybridization is resistant to RNase treatment in cell culture.

[0167] This example illustrates the use of anchored bridge probes (which hybridize with RNA in a biological sample and then subsequently with primary probes) in a polymer matrix providing for removal of cellular targets including the target RNA (e.g., RNase-insensitive methods to determine RNA in biological samples) according to certain embodiments of the invention. This example demonstrates the anchoring of bridge probes (with and without an anchoring agent), prepared according to Example 1, subsequent cellular component clearing, hybridization with MERFISH probes (e.g., primary probes) and imaging and compared to traditional MERFISH methods.VIZG019.PCT / US

[0168] In this example, a biological sample (e.g. U2OS cell line) was fixed by 4% paraformaldehyde (PFA) for 15 minutes at room temperature and then permeabilized by 70% ethanol overnight at 4°C. After washing the sample briefly with Sample Prep Wash Buffer (Vizgen, 20300001), the samples were either hybridized with a standard unmodified encoding probes (e.g., primary probe), or bridge probes with an acrydite moiety targeting 130 genes, diluted in Vizgen’s encoding probe hybridization buffer at a final concentration of 2 nanomole per probe (Vizgen 10400001) for two days at 37°C. Any unbound primary probes were removed with washing with Vizgen’s Formamide Wash Buffer (Vizgen, 20300002) at 47 °C for 30 minutes, twice. For one of the samples hybridized with the bridge probes, an anchoring agent (Vizgen pre -anchoring activator, (Vizgen, PN 20300113)) was added at 37°C for 1 hour, while the other sample was left untreated. The anchoring agents add acryloyl “tails’ to the backbone of DNA or RNA probes. Next, the biological sample was embedded in a polymer matrix using the Gel Embedding Premix (Vizgen 20300004), ammonium persulfate (Sigma, 09913-100G) and TEMED (N, N, N’, N’-tetramethylethylenediamine) (Sigma, T7024-25ME) from the MERSCOPE Sample Prep Kit (Vizgen, 10400012) wherein the acrydite moiety of the primary probe formed a covalent bond with the polyacrylamide gel. The gel embedded biological sample was treated with tissue clearing reagents consisting of 50uL of Protease K (NEB, P8107S) and 5mE of Clearing Premix (Vizgen 20300003) at 37C overnight to remove cellular components. The two samples hybridized with bridge probes were further treated with RNase A and H to degrade RNA, and then hybridized with primary probes (e.g., encoding probes) that bind to the target sequence of the bridge probes, followed by labeled secondary probes and imaging subsequently imaged on MERSCOPE for in situ gene expression profiling. See Figure 3, which shows RNA transcript counts per field of view (FOV) for the three different conditions tested.

[0169] Figure 4 shows a correlation analysis that was performed. The correlation analysis with bulk RNAseq data indicate that MERFISH measurement with bridge probe-mediated encoding probe hybridization is accurate and comparable to a standard MERFISH library hybridization. Fig. 4B reports a correlation analysis with bridge library with (Y axis) or without (Y axis) anchoring agent treatment. Fig. 5 depicts the spatial distribution of all genes in U2OS cells in MERFISFI multiplexed images.

[0170] This example demonstrates the anchored bridge probes (as a proxy for the target RNA in the biological sample) can be imaged using primary probes that hybridize to the bridge probes after RNase treatment providing an RNase insensitive method for imaging RNA transcripts in a biological sample.VIZG019.PCT / USIn other words, the biological sample was no longer sensitive to RNA degradation and was used for prolonged imaging, since the bridge probes were immobilized in the gel and not affected by RNA degradation processes in the sample.

[0171] Example 4. Use of modified paired split bridge probes with an anchor reagent for multiplexed imaging of nucleic acid analytes.

[0172] In this example, paired split bridge probes that arc complementary to the desired nucleic acid analytes are generated by techniques known in the art and / or as described in Example 1. The bridge probes of this current example, compared to Example 3, are split into two shorter paired probes, such that the binding of the MERFISH probe (e.g., primary nucleic acid probe) will happen when both split paired bridge probes are present in a sample. The split paired bridge probes are then modified with an anchoring agent and / or anchoring moiety (e.g., acrydite moiety) at a specific site on the probe, such that the anchoring agent can form a covalent bond with a gel, gel matrix, hydrogel, or other gel substance useful for the experiment. Using techniques known in the art, the modified split paired bridge probes are used to hybridize with the desired nucleic acid analytes in a biological specimen. The desired nucleic acid analytes can be RNA. In the next step, the means for creating a hydrogel matrix is introduced into the experimental setup and contemporaneously causing covalent bonds between the hydrogel matrix and the anchoring agent. The modified split pair bridge probes are now immobilized in the hydrogel matrix in the position where they were hybridized to the nucleic acid analytes. In the next step, the nucleic acid analytes as well as other cellular contents are removed chemically or enzymatically or both, leaving a non-cellular matrix to which the modified split pair bridge probes are still chemically7cross-linked. In the next step, MERFISH probes, designed by techniques known in the art comprising a target hybridization sequence and one or more read sequences hybridize to the modified split pair bridge probes are introduced into the experimental setup and allowed to hybridize according to the experimental design. To -analyze the experiment with a MERSCOPE, the signal from multiple rounds of readout probes is detected, according to standard MERFISH technique.

[0173] Example 5: Multiplexed imaging of 128 RNA species with bridge probe-mediated encoding probe hybridization is resistant to RNase treatment in mouse brain

[0174] This example illustrates the use of anchored bridge probes (which hybridize with RNA in the mouse brain tissue sample and then subsequently with primary probes) in a polymer matrix providingVIZG019.PCT / USfor removal of cellular targets including the target RNA (e.g., RNase-insensitive methods to determine RNA in biological samples) according to certain embodiments of the invention. This example demonstrates the anchoring of bridge probes (with and without an anchoring agent), prepared according to Example 1, subsequent cellular component clearing, hybridization with MERFISH probes (e.g., primary probes) and imaging and compared to traditional MERFISH methods.

[0175] In this example, a biological sample (e.g. mouse brain) was fixed by 4% paraformaldehyde (PFA) for 15 minutes at room temperature and then permeabilized by 70% ethanol overnight at 4°C. After washing the sample briefly with Sample Prep Wash Buffer (Vizgen, 20300001), the samples were either hybridized with a standard unmodified encoding probes, or bridge probes with an acrydite moiety targeting 128 genes, diluted in Vizgen’s encoding probe hybridization buffer at a final concentration of 2 nanomole per probe (Vizgen 10400001) for two days at 37°C. Any unbound primary probes were removed with washing with Vizgen’s Formamide Wash Buffer (Vizgen, 20300002) at 47’C for 30 minutes, twice. For the sample hybridized with the bridge probes, an anchoring agent (Vizgen pre-anchoring activator, (Vizgen, PN 20300113)) was added at 37°C for 1 hour. The anchoring agents add acryloyl “tails’ to the backbone of DNA or RNA probes. Next, the biological sample was embedded in a polymer matrix using the Gel Embedding Premix (Vizgen 20300004), ammonium persulfate (Sigma, 09913-100G) and TEMED (N, N, N’, N’-tetramethylethylenediamine) (Sigma, T7024-25ML) from the MERSCOPE Sample Prep Kit '(Vizgen, 10400012) wherein the acrydite moiety of the primary probe formed a covalent bond with the polyacrylamide gel. The gel embedded biological sample was treated with tissue clearing reagents consisting of 50uL of Protease K (NEB, P8107S) and 5mL of Clearing Premix (Vizgen 20300003) at 37C overnight to remove cellular components. The sample hybridized with bridge probes were further treated with RNase A and H to degrade RNA, and then hybridized with primary probes (e.g., encoding probes) that bind to the target sequence of the bridge probes, followed by labeled secondary probes and imaging subsequently imaged on MERSCOPE for in situ gene expression profiling The bridge probes convert the target gene into bridge probes anchored in a gel (“complementary target”, which is anchored in the polymer matrix), RNase-treated, and then hybridized with the matching primary probes (e.g., MERFISH probes) that have a target region reverse complementary (TRRC) to the anchored bridge probes. See Fig. 6A which shows a bar chart that summarizes the similarity of the MERFISH counts (RNA transcripts) per field of view (FOV) of the control method (e.g., no anchored bridge probes) and the RNase-insensitive techniques, according to the present invention (e.g., use ofVIZG019.PCT / USan anchored bridge probe to generate a complementary target that is then imaged using primary nucleic acid probes).

[0176] Correlation analysis with bulk RNAseq data indicate that MERFISH measurement with bridge probe-mediated primary probe hybridization is accurate and comparable to a standard MERFISH library hybridization. Fig. 6B shows the correlation plot of MERFISH counts / FOV on the Y axis and FPKM (fragments per kilobase per million reads) from bulk RNA sequencing data on the X axis. A correlation plot between the control (Y axis) and bridge probe experiments (X axis) of MERFISH counts / FOV also indicate the accuracy and comparability. See Figure 6B. Fig. 6C shows the spatial distribution of the 128 genes analyzed using the anchored bridge probes as a proxy for the genes in the mouse brain tissue sample, with four select genes highlighted on the right.

[0177] Example 6: Multiplexed imaging with bridge-mediated encoding probe hybridization of fresh frozen mouse heart and formalin-fixed, paraffin-embedded (FFPE) mouse liver.

[0178] This example illustrates the use of anchored bridge probes in two different protocols (“A” and “B”, See Figure 1) based on the tissue sample, wherein for the FFPE sample the RNA was first anchored in the polymer gel, cellular components cleared and then the bridge probes were added and anchored in a second polymer matrix.

[0179] In this example, multiplexed imaging with bridge probe-mediated encoding probe hybridization experiments of fresh frozen mouse heart and formalin-fixed, paraffin-embedded (FFPE) mouse liver were compared to control experiments (i.e., no anchored bridge probe). The control groups were hybridized with the standard 128-plex MERFISH library (VIZGEN) then imaged with the MERSCOPE (VIZGEN) instrument using standard techniques. The bridge probe-mediated modification experiments utilized the bridge probe-mediated gene library targeting 128 genes, according to Example 5 above, and then imaged on MERSCOPE.

[0180] In one experiment, fresh frozen mouse heart tissue samples were used, and the workflow performed according to “A” of Figure 1. In this example, a biological sample (e.g. mouse heart) was fixed by 4% paraformaldehyde (PFA) for 15 minutes at room temperature and then permeabilized by 70% ethanol overnight at 4°C. After washing the sample briefly with Sample Prep Wash Buffer (Vizgen, 20300001), the samples were either hybridized with a standard unmodified encoding probes, or bridge probes with an acrydite moiety targeting 128 genes, diluted in Vizgen’ s encoding probe hybridization buffer at a final concentration of 2 nanomole per probe (Vizgen 10400001) for two daysVIZG019. PCT / IJSat 37°C. Any unbound primary probes were removed with washing with Vizgen’s Formamide Wash Buffer (Vizgen, 20300002) at 47 ’C for 30 minutes, twice. For the sample hybridized with the bridge probes, an anchoring agent (Vizgen pre -anchoring activator, (Vizgen, PN 20300113)) was added at 37°C for 1 hour. The anchoring agents add acryloyl “tails’ to the backbone of DNA or RNA probes. Next, the biological sample was embedded in a polymer matrix using the Gel Embedding Premix (Vizgen 20300004), ammonium persulfate (Sigma, 09913-100G) and TEMED (N, N, N’, N’-tetramethylethylenediamine) (Sigma, T7024-25ML) from the MERSCOPE Sample Prep Kit (Vizgen, 10400012) wherein the acrydite moiety of the primary probe formed a covalent bond with the polyacrylamide gel. The gel embedded biological sample was treated with tissue clearing reagents consisting of 50uL of Protease K (NEB, P8107S) and 5mL of Clearing Premix (Vizgen 20300003) at 37C overnight to remove cellular components. The sample hybridized with bridge probes were further treated with RNase A and H to degrade RNA, and then hybridized with primary probes (e.g., encoding probes) that bind to the target sequence of the bridge probes, followed by labeled secondary probes and imaging subsequently imaged on MERSCOPE for in situ gene expression profiling. The bridge probes convert the target gene into bridge probes anchored in a gel (“complementary target”, which is anchored in the polymer matrix), RNase-treated, and then hybridized with the matching primary probes (e.g., MERFISH probes) that have a target region reverse complementary (TRRC) to the anchored bridge probes.

[0181] In a second experiment, FFPE mouse liver samples were used, and the workflow performed according to “B” of Figure 1. In this example, a biological sample (e.g. mouse liver) was deparaffinized with Deparaffinization Buffer (Vizgen 20300112) at 55°C for 5 minutes, 3 times, and then rehydrated with 100% ethanol for 2 minutes, 90% ethanol for 2 minutes, 70% ethanol for 2 minutes. The sample was then decrosslinked by Decrosslinking Buffer (Vizgen, 20300115) at 90°C for 15 min, incubated with Conditioning Buffer (Vizgen 20300116) at 37°C for 30 min, followed with Pre -Anchoring Reaction Buffer containing lOOpl Conditioning Buffer (Vizgen 20300116), 5 l Pre -Anchoring Activator (Vizgen, 20300113) and 5pl RNase inhibitor (NEB, M0314L) at 37°C for 2 hours. The sample was then incubated with Anchoring Buffer (PN 20300117) at 37°C overnight. Next, the biological sample was embedded in a polymer matrix using the Gel Embedding Premix (Vizgen 20300004), ammonium persulfate (Sigma, 09913-100G) and TEMED (N, N, N’, N’-tetramethylethylenediamine) (Sigma, T7024-25ME) from the MERSCOPE Sample Prep Kit (Vizgen, 10400012). The gel embedded biological sample was treated with tissue clearing reagents consistingVIZG019.PCT / USof 50uL of Protease K (NEB, P8107S) and 5mL of Clearing Premix (Vizgen 20300003) at 37C overnight to remove cellular components. After washing the sample briefly with Sample Prep Wash Buffer (Vizgen, 20300001), the samples were either hybridized with a standard unmodified encoding probes, or bridge probes with an acrydite moiety targeting 128 genes, diluted in Vizgen’s encoding probe hybridization buffer at a final concentration of 2 nanomole per probe (Vizgen 10400001) for two days at 37 °C. Any unbound primary probes were removed with washing with Vizgen’s Formamide Wash Buffer (Vizgen, 20300002) at 47“C for 30 minutes, twice. For the sample hybridized with the bridge probes, an anchoring agent (Vizgen pre -anchoring activator, (Vizgen, PN 20300113)) was added at 37 " C for 1 hour. The anchoring agents add acryloyl “tails’ to die backbone of DNA or RNA probes. The sample was gel embedded again, and further treated with RNase A and H to degrade RNA, and tiien hybridized with primary probes (e.g., encoding probes) that bind to the target sequence of the bridge probes, followed by labeled secondary probes and imaging subsequently imaged on MERSCOPE for in situ gene expression profiling. The bridge probes convert the target gene into bridge probes anchored in a gel (“complementary target”, which is anchored in die polymer matrix), RNase-treated, and then hybridized widi the matching primary probes (e.g., MERFISH probes) that have a target region reverse complementary (TRRC) to the anchored bridge probes.

[0182] Figure 7 shows that the MERFISFI (transcripts) counts per field of view (FOV) were similar between the control experiment and the bridge probe-mediated modification experiment in both the fresh frozen mouse heart (A) and FFPE mouse liver (B) studies, which demonstrated the RNase-insensitivity ot an embodiment ot the present invention.

Claims

1. VIZG019. PCT / IJSCLAIMSWhat is claimed is:

1. A method for anchoring within a polymer matrix a complementary target of a distinct nucleic acid species and clearing cellular components comprising:a. contacting a biological sample comprising the distinct nucleic acid species with a bridge probe and an anchoring agent, wherein the bridge probe comprises an acrydite moiety and hybridizes with the distinct nucleic acid species and the anchoring agent forms a covalent bond with the bridge probe, wherein when hybridized the bridge probe provides a complementary target of the distinct nucleic acid species;b. embedding the biological sample in a polymer matrix wherein foe complementary target of the distinct nucleic acid species and foe anchoring agent each form a covalent bond with the polymer matrix; andc. clearing cellular components from the polymer matrix wherein the complementary target of the distinct nucleic acid species remain anchored in the polymer matrix to form a matrix anchored complementary target sample.

2. The method of claim 1, further comprising adding RNase to foe matrix anchored complementary target sample and clearing foe distinct nucleic acid species from the polymer matrix.

3. The method of claim 1, wherein the anchoring agent is an alkylating agent.

4. The method of claim 3, wherein foe alkylating agent is selected from the group consisting of Altretamine, Bendamustine, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine, Ifosfamide, Lomustine, Mechlorefoamine, Melphalan, Oxaliplatin, Temozolomide, Thiotepa and Trabectedin.

5. The method of claim 1, wherein foe anchoring agent and the bridge probe each comprise an acrydite moiety that covalently binds the polymer matrix.

6. The method of claim 1, wherein the anchoring agent is an alkylating agent derivatized with an acrydite moiety.

7. The method of claim 1, wherein the distinct nucleic acid species is RNA.VIZG019.PCT / US8. The method of claim 1, wherein the distinct nucleic acid species is DNA.

9. The method of claim 1, wherein the bridge probe comprises a target sequence that hybridizes to the distinct nucleic acid species and an acrydite moiety that covalently binds the polymer matrix.

10. The method of claim 1, further comprising imaging the anchored complementary target of the distinct nucleic acid species by contacting the anchored complementary target with a primary nucleic acid probe.

11. The method of claim 10, wherein the primary nucleic acid probe comprises a target sequence that hybridizes to the complementary target and one or more read sequences.

12. The method of claim 10, wherein the primary nucleic acid probes are single molecule (sm)FISH probes or multiplexed error robust fluorescence in situ hybridization (MERFISH) probes.

13. The method of claim 11, further comprising determining the one or more read sequences based on contacting the primary nucleic acid probe with a plurality of secondary nucleic acid probes comprising a recognition sequence that hybridizes to the read sequence of the primary nucleic acid probe.

14. The method ot claim 10, further comprising imaging using multiplexed fluorescence in situ hybridization comprising one or more sequential steps of adding a plurality of secondary nucleic acid probes comprising a label moiety that hybridize to the primary nucleic acid probes.

15. The method of claim 10, further comprising imaging using multiplexed error robust fluorescence in situ hybridization (MERFISH) probes wherein the primary nucleic acid probes are MERFISH probes.

16. The method of claim 1, wherein step a) comprises contacting the biological sample comprising a plurality of distinct RNA species with a plurality of nucleic acid bridge probe pools each of the bridge probes comprising a target sequence and an acrydite moiety that covalently binds to a polymer matrix, wherein when hybridized the bridge probe pools provide a complementary target of the distinct nucleic acid species.

17. The method of claim 16, further comprising contacting the anchored bridge probes with a plurality of primary nucleic acid probe pools, each of the primary nucleic acid probes comprising a bridge probe target sequence and one or more read sequences, wherein each pool of primary nucleic acidVIZG019.PCT / USprobes hybridize to a distinct complementary target of the distinct RNA species and each pool of primary probes encode a N-bit code that was assigned to each distinct RNA species, wherein each assigned N-bit code is a valid codeword with a Hamming distance of 2 or more between valid codewords.

18. The method of claim 17, wherein each of the read sequences correspond to a bit value of 1 for the codewords assigned to the distinct RNA species.

19. The method of claim 17, further comprising contacting the primary nucleic acid probes with a plurality of readout probes comprising a fluorescent label, wherein the readout probes hybridize to the read sequences of the primary nucleic acid probes; imaging the readout probes bound to the primary nucleic acid probes; and, repeating steps in one or more sequential hybridization and imaging rounds until all N positions in the N-bit code have been imaged to provide an imaged codeword corresponding to each distinct RNA species in a spatial organization.

20. The method of claim 17, wherein the valid codewords have a Hamming distance equal to or greater than 4 between each of the valid codewords.

21. The method of claim 17, wherein the N-bit code has a Hamming weight of 4.

22. The method of claim 19, wherein the imaged codeword is matched to a valid codeword assigned to a distinct RNA species.

23. The method of clam 19, wherein the Hamming distance is 4 or greater and the imaged codewords are corrected and matched to a valid codeword or discarded.

24. The method of clam 19, wherein the Hamming distance is 4 or greater and the imaged codewords are matched to a valid codeword or discarded.

25. The method of claim 19, wherein the RNA species is a RNA transcript.

26. The method of claim 19, comprising determining the spatial organization of the transcriptome from a single cell.

27. The method of claim 17, wherein each primary nucleic acid probe pool comprises at least 10 different primary nucleic acid probes.VIZG019.PCT / US28. The method of claim 17, wherein each primary nucleic acid probe pool comprises at least four distinct read sequences.

29. The method of claim 17, wherein each primary nucleic acid probe pool comprises four distinct read sequences.

30. The method of claim 17, wherein each primary nucleic acid probe pool comprises at least eight distinct read sequences.

31. The method of claim 17, wherein the primary nucleic acid probes comprise a target sequence, with an average length of between 10 and 200 nucleotides, that hybridize the distinct RNA species.

32. The method of claim 17, wherein each primary nucleic acid probe pool comprises at least 10 different target sequences.

33. The method of claim 19, wherein after each hybridization and imaging round the fluorescent read out probe is quenched to inactivate.

34. The method of claim 19, wherein after each hybridization and imaging round the fluorescent read out probe is inactivated by chemically or enzymatically cleaving the fluorescent label from the readout probe.

35. The method of claim 17, wherein the N-bit code comprises at least a 16-bit code.

36. The method of claim 19, wherein the plurality of fluorescent readout probes comprise at least two distinct fluorescent labels.

37. The method of claim 19, wherein the plurality of fluorescent readout probes comprise at least three distinct fluorescent labels.

38. The method of claim 19, wherein the spatial organization of the distinct RNA species is imaged in 2 dimensions.

39. The method of claim 19, wherein the spatial organization of the distinct RNA species is imaged in 3 dimensions.

40. The method of claim 19, further comprising determining abundance for the distinct RNA species.

41. The method of claim 1, further comprising anchoring the distinct nucleic acid species in a polymer matrix prior to step a).VIZG019.PCT / US42. The method of claim 41, wherein anchoring the distinct nucleic acid species comprises contacting the biological sample with a first and / or second anchoring agent, wherein the first anchoring agent forms a covalent bond with the distinct nucleic acid species and the second anchoring agent comprises an oligonucleotide that hybridizes with the distinct nucleic acid species.

43. The method of claim 42, wherein the first anchoring agent is an alkylating agent.

44. The method of claim 43, wherein the alkylating agent is selected from the group consisting of Altretamine, Bendamustine, Busulfan, Carboplatm, Carmustine, Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine, Ifosfamide, Lomustine, Mechlorethamine, Melphalan, Oxaliplatin, Temozolomide, Thiotepa and Trabectedin.

45. The method of claim 42, wherein the second anchoring agent comprises alternating dT and locked d'l' portions that hybridizes with the distinct nucleic acid species.

46. The method of claim 42, wherein the second anchoring agent comprises a poly-dT portion that hybridizes to the distinct nucleic acid species.

47. The method of claim 42, wherein the first anchoring agent and the second anchoring agent each comprise an acrydite moiety that covalently binds the polymer matrix.

48. The method of claim 42, wherein the first anchoring agent is an alkylating agent derivatized with an acrydite moiety.

49. The method of claim 42, wherein the second anchoring agent comprises a poly-dT portion that hybridizes to the distinct nucleic acid species and an acrydite moiety that covalently binds the polymer matrix.

50. The method of claim 42, wherein the distinct nucleic acid species is RNA.

51. The method of claim 42, wherein the distinct nucleic acid species is DNA.

52. The method of claim 16, further comprising imaging the complementary target of the distinct nucleic acid species by a method comprising:d) contacting the anchored bridge probes with a plurality of primary nucleic acid probe pools each of the primary nucleic acid probes comprising a bridge probe target sequence and one or more read sequences, wherein each pool of primary nucleic acid probes hybridizesVIZG019.PCT / USto a distinct complementary target of the distinct RNA species and each pool of primary probes encode a N-bit code that was assigned to each distinct RNA species, wherein each assigned N-bit code is a valid codeword with a Hamming distance of 2 between valid codewords;e) contacting the bound primary nucleic acid probes with a plurality of fluorescent readout probes that hybridize to the read sequences of the primary nucleic acid probes; f) imaging the readout probes bound to the primary nucleic acid probes; and,g) repeating steps e) and f) in one or more sequential hybridization and imaging rounds until all N positions in the N- bit code have been imaged providing an imaged bit code corresponding to each of the distinct nucleic acid species in a spatial organization.

53. A method of imaging a complementary target of a distinct nucleic acid species and clearing cellular components, the method comprising:a) contacting a biological sample comprising the distinct nucleic acid species with a bridge probe and an anchoring agent, wherein the bridge probe comprises an acrydite moiety and hybridizes with the distinct nucleic acid species and the anchoring agent forms a covalent bond with the bridge probe, wherein when hybridized with the distinct nucleic acid species the bridge probe provides a complementary target for the distinct nucleic acid species;b) embedding the biological sample in a polymer matrix wherein the complementary target of the distinct nucleic acid species and the anchoring agent each form a covalent bond with the polymer matrix;c) clearing cellular components from the polymer matrix wherein the complementary target of the distinct nucleic acid species remains anchored in the polymer matrix to form a matrix anchored complementary target;d) contacting the anchored complementary target with a primary nucleic acid probe comprising a bridge probe hybridization sequence and one or more read sequences; and,VIZG019.PCT / USe) contacting the primary nucleic acid probe with a plurality of fluorescent readout probes that hybridize to the one or more read sequences of the primary nucleic acid probes and imaging the complementary target of the distinct nucleic acid species.

54. The method of claim 53, further comprising adding RNase to the matrix anchored complementary target sample and clearing the distinct nucleic acid species from the polymer matrix.

55. The method of claim 53, wherein the anchoring agent is an alkylating agent.

56. The method of claim 55, wherein the alkylating agent is selected from the group consisting of Altretamine, Bendamustine, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine, Ifosfamide, Tomustine, Mechlorethamine, Melphalan, Oxaliplatin, Temozolomide, Thiotepa and Trabectedin.

57. The method of claim 53, wherein the anchoring agent and the bridge probe each comprise an acrydite moiety that covalently binds the polymer matrix.

58. The method of claim 53, wherein the anchoring agent is an alkylating agent derivatized with an acrydite moiety.

59. The method of claim 53, wherein the distinct nucleic acid species is RNA.

60. The method of claim 53, wherein the distinct nucleic acid species is DNA.

61. The method of claim 53, wherein the primary nucleic acid probe comprises a target sequence that hybridizes to the bridge probe, one or more read sequences and an acrydite moiety that covalently binds the polymer matrix.

62. The method of claim 53, wherein the primary nucleic acid probes are single molecule (sm)FISH probes or multiplexed error robust fluorescence in situ hybridization (MERFISH) probes.

63. The method of claim 53, further comprising determining read sequences based on contacting the primary nucleic acid probe with a plurality of secondary nucleic acid probes comprising a recognition sequence that hybridizes to the read sequence of the primary nucleic acid probe.

64. The method of claim 53, wherein step e) is repeated one or more times comprising one or more sequential steps of adding a plurality of secondary nucleic acid probes comprising a label moiety that hybridize to the anchored primary nucleic acid probes.VIZG019.PCT / US65. The method of claim 64, further comprising imaging using multiplexed error robust fluorescence in situ hybridization (MERFISH) probes wherein the anchored primary nucleic acid probes are MERFISH probes anchored in the polymer matrix.

66. The method of claim 53, wherein the biological sample comprises a plurality of distinct RNA species and step a) comprises contacting the biological sample with a plurality of nucleic acid bridge probe pools and wherein each of the bridge probes comprises a target sequence and an acrydite moiety that covalently binds to a polymer matrix, and when hybridized the bridge probe pools provide a complementary target of the distinct nucleic acid species.

67. The method of claim 66, further comprising contacting the anchored bridge probes with a plurality of primary nucleic acid probe pools, each of the primary nucleic acid probes comprising a bridge probe target sequence and one or more read sequences, wherein each pool of primary nucleic acid probes hybridizes to a distinct complementary target of the distinct RNA species and each pool of primary nucleic acid probes encodes a different N-bit code assigned to each distinct RNA species, wherein each assigned N-bit code is a valid codeword with a Hamming distance of 2 between valid codewords.

68. The method of claim 67, wherein each of the read sequences corresponds to a bit value of 1 for the valid codewords assigned to the distinct RNA species.

69. The method of claim 67, further comprising contacting the primary nucleic acid probes with a plurality of readout probes comprising a fluorescent label, wherein the readout probes hybridize to the read sequences of the primary nucleic acid probes; imaging the readout probes bound to the primary nucleic acid probes; and, repeating steps in one or more sequential hybridization and imaging rounds until all N positions in the N-bit code have been imaged to provide an imaged codeword corresponding to each distinct RNA species in a spatial organization.

70. The method of claim 67, wherein the valid codewords have a Hamming distance equal to or greater than 4 between each of the valid codewords.

71. The method of claim 67, wherein the N-bit code has a Hamming weight of 4.

72. The method of claim 69, wherein each imaged codeword is matched to a valid codeword assigned to a distinct RNA species.VIZG019.PCT / US73. The method of clam 69, wherein the Hamming distance is 4 or greater and the imaged codewords are corrected and matched to a valid codeword or discarded.

74. The method of clam 69, wherein the Hamming distance is 4 or greater and the imaged codewords are matched to a valid codeword or discarded.

75. The method of claim 69, wherein the RNA species is a RNA transcript.

76. The method of claim 69, further comprising determining the spatial organization of the transcriptome from a single cell.

77. The method of claim 67, wherein each primary nucleic acid probe pool comprises at least 10 different primary nucleic acid probes.

78. The method of claim 67, wherein each primary nucleic acid probe pool comprises at least four distinct read sequences.

79. The method of claim 67, wherein each primary nucleic acid probe pool comprises four distinct read sequences.

80. The method of claim 67, wherein each primary nucleic acid probe pool comprises at least eight distinct read sequences.

81. The method of claim 67, wherein the primary nucleic acid probes comprise a target sequence, with an average length of between 10 and 200 nucleotides, that hybridize to the distinct RNA species.

82. The method of claim 67, wherein each primary nucleic acid probe pool comprises at least 10 different target sequences.

83. The method of claim 69, wherein after each hybridization and imaging round the fluorescent read out probe is quenched to inactivate.

84. The method of claim 69, wherein after each hybridization and imaging round the fluorescent read out probe is inactivated by chemically or enzymatically cleaving the fluorescent label from the readout probe.

85. The method of claim 67, wherein the N-bit code comprises at least a 16-bit code.

86. The method of claim 69, wherein the plurality of fluorescent readout probes comprise at least two distinct fluorescent labels.VIZG019.PCT / US87. The method of claim 69, wherein the plurality of fluorescent readout probes comprise at least three distinct fluorescent labels.

88. The method of claim 69, wherein the spatial organization of the distinct RNA species is imaged in 2 dimensions.

89. The method of claim 69, wherein the spatial organization of the distinct RNA species is imaged in 3 dimensions.

90. The method of claim 69, further comprising determining abundance for the distinct RNA species.

91. The method of claim 53, further comprising anchoring the distinct nucleic acid species in a polymer matrix prior to step a).

92. The method of claim 91, wherein anchoring the distinct nucleic acid species comprises contacting the biological sample with a first and / or second anchoring agent, wherein the first anchoring agent forms a covalent bond with the distinct nucleic acid species and the second anchoring agent comprises an oligonucleotide that hybridizes with the distinct nucleic acid species.

93. The method of claim 92, wherein the first anchoring agent is an alkylating agent.

94. The method of claim 93, wherein the alkylating agent is selected from the group consisting of Altretamine, Bendamustine, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine, Ifosfamide, Lomustine, Mechlorethamine, Melphalan, Oxaliplatin, Temozolomide, Thiotepa and Trabectedin.

95. The method of claim 92, wherein the second anchoring agent comprises alternating dT and locked dT portions that hybridizes with the distinct nucleic acid species.

96. The method of claim 92, wherein the second anchoring agent comprises a poly-dT portion that hybridizes to the distinct nucleic acid species.

97. The method of claim 92, wherein the first anchoring agent and the second anchoring agent each comprise an acrydite moiety that covalently binds the polymer matrix.

98. The method of claim 42, wherein the first anchoring agent is an alkylating agent derivatized with an acrydite moiety.VIZG019.PCT / US99. The method of claim 92, wherein the second anchoring agent comprises a poly-dT portion that hybridizes to the distinct nucleic acid species and an acrydite moiety that covalently binds the polymer matrix.

100. The method of claim 92, wherein the distinct nucleic acid species is RNA.

101. The method of claim 92, wherein the distinct nucleic acid species is DNA.

102. A method for generating and anchoring within a polymer matrix a complementary target of a plurality of distinct nucleic acid species comprising:a. contacting a sample comprising a plurality of distinct nucleic acid species with a plurality of nucleic acid bridge probe pools comprising bridge probes, each bridge probe comprising a target sequence and a moiety that covalently binds to a polymer matrix, wherein when hybridized the bridge probe pools provide a complementary target of the distinct nucleic acid species;b. polymerizing the polymer matrix within the sample;c. anchoring the complementary target of the distinct nucleic acid species to the polymer matrix wherein the bridge probes covalently bind the polymer matrix; and,d. clearing cellular components from the polymer matrix wherein the complementary target of the distinct nucleic acid species remain anchored in the polymer matrix.

103. The method of claim 102, further comprising imaging the complementary target of the distinct nucleic acid species comprising:a. contacting the bridge probes with a plurality of primary nucleic acid probe pools comprising primary nucleic acid probes, each primary nucleic acid probe comprising a bridge probe target sequence and one or more read sequences, wherein each pool of primary nucleic acid probes hybridize to a distinct complementary target of the distinct nucleic acid species and each pool of probes encode a N-bit code that was assigned to each distinct nucleic acid species;b. contacting the hybridized primary nucleic acid probes with a plurality of fluorescent readout probes that hybridize to the read sequences of the primary nucleic acid probes;VIZG019.PCT / USc. imaging the readout probes bound to the primary nucleic acid probes; and,d. repeating steps f) and g) in one or more sequential hybridization and imaging rounds until all N positions in the N-bit code have been imaged providing an imaged bit code corresponding to each of the distinct nucleic acid species in a spatial organization.

104. The method of claim 103, wherein the target sequences bridge two bridge probes when hybridized.

105. The method of claim 102, wherein the bridge probe is DNA.

106. The method of claim 102, wherein the plurality of distinct nucleic acid species is RNA.

107. The method of claim 102, wherein the moiety that covalently binds to a polymer matrix is an acrydite moiety.

108. The method of claim 102, further comprising adding RNase to the matrix anchored complementary target sample and clearing the distinct nucleic acid species from the polymer matrix.

109. The method of claim 102, wherein step a) further comprises contacting the biological sample with an anchoring agent, wherein the bridge probe comprises an acrydite moiety and hybridizes with the distinct nucleic acid species and the anchoring agent forms a covalent bond with the bridge probe.

110. The method of claim 109, wherein the anchoring agent is an alkylating agent.

111. The method ot claim 110, wherein the alkylating agent is selected from the group consisting of Altretamine, Bendamustine, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine, Ifosfamide, Lomus fine, Mechlorethamine, Melphalan, Oxaliplatin, Temozolomide, Thiotepa and Trabectedin.

112. The method of claim 109, wherein the anchoring agent and the bridge probe each comprise an acrydite moiety that covalently binds the polymer matrix.

113. The method of claim 109, wherein the anchoring agent is an alkylating agent derivatized with an acrydite moiety.VIZG019.PCT / US114. The method of claim 103, wherein each assigned N-bit code is a valid codeword with a Hamming distance of 2 between valid codewords.

115. The method of claim 103, wherein each of the read sequences correspond to a bit value of 1 for the codewords assigned to the distinct RNA species.

116. The method of claim 103, further comprising contacting the primary nucleic acid probes with a plurality of readout probes comprising a fluorescent label, wherein the readout probes hybridize to the read sequences of the primary nucleic acid probes; imaging the readout probes bound to the primary nucleic acid probes; and, repeating steps in one or more sequential hybridization and imaging rounds until all N positions in the N-bit code have been imaged providing an imaged codeword corresponding to each distinct RNA species in a spatial organization.

117. The method ot claim 114, wherein the valid codewords have a Hamming distance equal to or greater than 4 between each of the valid codewords.

118. The method of claim 103, wherein the N-bit code has a Hamming weight of 4.

119. The method of claim 103, wherein the imaged codeword is matched to a valid codeword assigned to a distinct RNA species.

120. The method of clam 103, wherein the Hamming distance is 4 or greater and the imaged codewords are corrected and matched to a valid codeword or discarded.

121. The method of clam 103, wherein the Hamming distance is 4 or greater and the imaged codewords are matched to a valid codeword or discarded122. The method of claim 116, wherein the RNA species is a RNA transcript.

123. The method of claim 103, comprising determining the spatial organization of die transcriptome from a single cell.

124. The method of claim 103, wherein each primary nucleic acid probe pool comprises at least 10 different primary nucleic acid probes.

125. The method of claim 103, wherein each primary nucleic acid probe pool comprises at least four distinct read sequences.VIZG019.PCT / US126. The method of claim 103, wherein each primary nucleic acid probe pool comprises four distinct read sequences.

127. The method of claim 103, wherein each primary nucleic acid probe pool comprises at least eight distinct read sequences.

128. The method of claim 103, wherein the primary nucleic acid probes comprise a target sequence, with an average length of between 10 and 200 nucleotides, that hybridize the distinct RNA species.

129. The method of claim 103, wherein each primary nucleic acid probe pool comprises at least 10 different target sequences.

130. The method of claim 103, wherein after each hybridization and imaging round the fluorescent readout probe is quenched to inactivate.

131. The method of claim 103, wherein after each hybridization and imaging round the fluorescent readout probe is inactivated by chemically or enzymatically cleaving the fluorescent label from the readout probe.

132. The method ot claim 103, wherein the N-bit code comprises at least a 16-bit code.

133. The method of claim 103, wherein the plurality of fluorescent readout probes comprise at least two distinct fluorescent labels.

134. The method of claim 103, wherein the plurality of fluorescent readout probes comprise at least three distinct fluorescent labels.

135. The method of claim 116, wherein the spatial organization of the distinct RNA species is imaged in 2 dimensions.

136. The method of claim 116, wherein the spatial organization of the distinct RNA species is imaged in 3 dimensions.

137. The method of claim 116, further comprising determining abundance for the distinct RNA species.

138. The method of claim 102, further comprising anchoring the distinct nucleic acid species in a polymer matrix prior to step a).VIZG019.PCT / US139. The method of claim 138, wherein anchoring the distinct nucleic acid species comprises contacting the biological sample with a first and / or second anchoring agent, wherein the first anchoring agent forms a covalent bond with the distinct nucleic acid species and the second anchoring agent comprises an oligonucleotide that hybridizes with the distinct nucleic acid species.

140. The method of claim 139, wherein the first anchoring agent is an alkylating agent.

141. The method of claim 140, wherein the alkylating agent is selected from the group consisting of Altretamine, Bendamustine, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine, Ifosfamide, Lomustine, Mechlorethamine, Melphalan, Oxaliplatin, Temozolomide, Thiotepa and Trabectedin.

142. The method of claim 139, wherein the second anchoring agent comprises alternating dT and locked dT portions that hybridizes with the distinct nucleic acid species.

143. The method of claim 139, wherein the second anchoring agent comprises a poly-dT portion that hybridizes to the distinct nucleic acid species.

144. The method of claim 139, wherein the first anchoring agent and the second anchoring agent each comprise an acrydite moiety that covalently binds the polymer matrix.

145. The method of claim 139, wherein the first anchoring agent is an alkylating agent derivatized with an acrydite moiety.

146. The method of claim 139, wherein the second anchoring agent comprises a poly-dT portion that hybridizes to the distinct nucleic acid species and an acrydite moiety that covalently binds the polymer matrix.

147. The method of claim 139, wherein the distinct nucleic acid species is RNA.

148. The method of claim 139, wherein the distinct nucleic acid species is DNA.

149. A method for anchoring within a polymer matrix a complementary target of a distinct nucleic acid species and clearing cellular components comprising:a. contacting a biological sample with a first and / or second anchoring agent, wherein the first anchoring agent forms a covalent bond with the distinct nucleic acid species and the secondVIZG019.PCT / USanchoring agent comprises an oligonucleotide that hybridizes with the distinct nucleic acid species and wherein the first anchoring agent and the second anchoring agent each comprise an acrydite moiety that covalently binds to the polymer matrix;b. embedding the biological sample in a polymer matrix wherein the first and second anchoring agents each form a covalent bond with the polymer matrix;c. clearing the cellular components from the polymer matrix wherein the distinct nucleic acid species remains anchored in the polymer matrix;d. contacting the anchored distinct nucleic acid species with a bridge probe and the first anchoring agent, wherein the bridge probe comprises an acrydite moiety and hybridizes with the distinct nucleic acid species and the first anchoring agent forms a covalent bond with the bridge probe, wherein when hybridized the bridge probe provides a complementary target of the distinct nucleic acid species;e. embedding the bridge probe sample in a polymer matrix wherein the complementary target of the distinct nucleic acid species and the anchoring agent each form a covalent bond with the polymer matrix to form a matrix anchored complementary target sample.

150. The method of claim 149, further comprising adding RNase to the matrix anchored complementary target sample and clearing the distinct nucleic acid species from the polymer matrix.

151. The method of claim 149, wherein the first anchoring agent is an alkylating agent.

152. The method of claim 151, wherein the alkylating agent is selected from the group consisting of Altretamine, Bendamustine, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine, Ifosfamide, Lomustine, Mechlorethamine, Melphalan, Oxaliplatin, Temozolomide, Thiotepa and Trabectedin.

153. The method of claim 149, wherein the first anchoring agent and the bridge probe each comprise an acrydite moiety that covalently binds the polymer matrix.

154. The method of claim 149, wherein the first anchoring agent is an alkylating agent derivatized with an acrydite moiety.

155. The method of claim 149, wherein the distinct nucleic acid species is RNA.VIZG019.PCT / US156. The method of claim 149, wherein the distinct nucleic acid species is DNA.

157. The method of claim 149, wherein the bridge probe comprises a target sequence that hybridizes to the distinct nucleic acid species and an acrydite moiety that covalently binds die polymer matrix.

158. The method of claim 149, further comprising imaging the anchored complementary target of the distinct nucleic acid species by contacting the anchored complementary target with a primary nucleic acid probe.

159. The method of claim 158, wherein the primary nucleic acid probe comprises a target sequence that hybridizes to the complementary target and one or more read sequences.

160. The method of claim 159, wherein the primary nucleic acid probes are single molecule (sm)FISH probes or multiplexed error robust fluorescence in situ hybridization (MERFISH) probes.

161. The method of claim 159, further comprising determining the one or more read sequences based on contacting the primary nucleic acid probe with a plurality of secondary nucleic acid probes comprising a recognition sequence that hybridizes to the read sequence of the primary nucleic acid probe.

162. The method of claim 158, further comprising imaging using multiplexed fluorescence in situ hybridization comprising one or more sequential steps of adding a plurality of secondary nucleic acid probes comprising a label moiety that hybridize to the primary nucleic acid probes.

163. The method of claim 158, further comprising imaging using multiplexed error robust fluorescence in situ hybridization (MERFISH) probes wherein the primary nucleic acid probes are MERFISH probes.

164. The method of claim 149, wherein the anchored distinct nucleic acid species comprise distinct RNA species and step d) comprises contacting the anchored distinct RNA species with a plurality of nucleic acid bridge probe pools each of the bridge probes comprising a target sequence and an acrydite moiety that covalently binds to a polymer matrix, wherein when hybridized the bridge probe pools provide a complementary target of the distinct RNA species.

165. The method of claim 164, further comprising contacting the anchored bridge probes with a plurality of primary nucleic acid probe pools, each of the primary nucleic acid probes comprisingVIZG019.PCT / USa bridge probe target sequence and one or more read sequences, wherein each pool of primary nucleic acid probes hybridize to a distinct complementary target of the distinct RNA species and each pool of primary probes encode a different N-bit code assigned to each distinct RNA species, wherein each assigned N-bit code is a valid codeword with a Hamming distance of 2 between valid codewords.

166. The method of claim 165, wherein each of the read sequences correspond to a bit value of 1 for the codewords assigned to the distinct RNA species.

167. The method of claim 165, further comprising contacting the primary nucleic acid probes with a plurality of readout probes comprising a fluorescent label, wherein the readout probes hybridize to the read sequences of the primary nucleic acid probes; imaging the readout probes bound to the primary nucleic acid probes; and, repeating steps in one or more sequential hybridization and imaging rounds until all N positions in the N-bit code have been imaged providing an imaged codeword corresponding to each distinct RNA species in a spatial organization.

168. The method of claim 165, wherein the valid codewords have a Hamming distance equal to or greater than 4 between each of the valid codewords.

169. The method of claim 165, wherein the N-bit code has a Hamming ’eight of 4.

170. The method of claim 167, wherein the imaged codeword is matched to a valid codeword assigned to a distinct RNA species.

171. The method of clam 167, wherein the Hamming distance is 4 or greater and the imaged codewords are corrected and matched to a valid codeword or discarded.

172. The method of clam 167, wherein the Hamming distance is 4 or greater and the imaged codewords are matched to a valid codeword or discarded173. The method of claim 167, wherein each distinct RNA species is a RNA transcript.

174. The method of claim 167, comprising determining the spatial organization of the transcriptome from a single cell.

175. The method of claim 165, wherein each primary nucleic acid probe pool comprises at least 10 different primary nucleic acid probes.VIZG019.PCT / US176. The method of claim 165, wherein each primary nucleic acid probe pool comprises at least four distinct read sequences.

177. The method of claim 165, wherein each primary nucleic acid probe pool comprises four distinct read sequences.

178. The method of claim 165, wherein each primary nucleic acid probe pool comprises at least eight distinct read sequences.

179. The method of claim 165, wherein the primary nucleic acid probes comprise a target sequence, with an average length of between 10 and 200 nucleotides, that hybridize the distinct RNA species.

180. The method of claim 165, wherein each primary nucleic acid probe pool comprises at least 10 different target sequences.

181. The method of claim 167, wherein after each hybridization and imaging round the fluorescent read out probe is quenched to inactivate.

182. The method of claim 167, wherein after each hybridization and imaging round the fluorescent read out probe is inactivated by chemically or enzymatically cleaving the fluorescent label from the readout probe.

183. The method of claim 165, wherein the N-bit code comprises at least a 16-bit code.

184. The method of claim 167, wherein the plurality of fluorescent readout probes comprise at least two distinct fluorescent labels.

185. The method of claim 167, wherein the plurality of fluorescent readout probes comprise at least three distinct fluorescent labels.

186. The method of claim 167, wherein the spatial organization of the distinct RNA species is imaged in 2 dimensions.

187. The method of claim 167, wherein the spatial organization of the distinct RNA species is imaged in 3 dimensions.

188. The method of claim 167, further comprising determining abundance for the distinct RNA species.VIZG019.PCT / US189. The method of claim 149, wherein the second anchoring agent comprises alternating dT and locked dT portions that hybridizes with the distinct nucleic acid species.

190. The method of claim 149, wherein the second anchoring agent comprises a poly-dT portion that hybridizes to the distinct nucleic acid species.

191. The method of claim 149, wherein the first anchoring agent and the second anchoring agent each comprise an acrydite moiety that covalently binds the polymer matrix.

192. The method of claim 149, wherein the second anchoring agent comprises a poly-dT portion that hybridizes to the distinct nucleic acid species and an acrydite moiety that covalently binds the polymer matrix.

193. The method of any one of claims 1-192, wherein the biological sample has been deparaffinized and rehydrated prior to step (a).

194. The method of any one of claims 1-192, wherein the polymer matrix comprises polyacrylamide.

195. The method of any one of claims 1-192, wherein the cellular components comprise proteins, lipids, DNA, RNA, or extracellular matrix.

196. The method of any one of claims 1-192, wherein clearing the cellular components comprises removing or degrading proteins from the biological sample.

197. The method of any one of claims 1-192, wherein clearing the cellular components comprises removing or degrading lipids from the biological sample.

198. The method of any one of claims 1-192, wherein clearing the cellular components comprises removing or degrading target RNA from the biological sample.

199. The method of any one of claims 1-192, wherein clearing the cellular components comprises removing or degrading extracellular matrix from the biological sample.

200. The method of any one of claims 1-192, wherein clearing the cellular components comprises exposing the biological sample to an enzyme able to degrade a protein.

201. The method of any one of claims 1-192, wherein clearing the cellular components comprises exposing the biological sample to an enzyme able to degrade DNA.VIZG019.PCT / US202. The method of any one of claims 1-192, wherein clearing the cellular components comprises exposing the biological sample to an enzyme able to degrade RNA.

203. The method of any one of claims 1-192, wherein clearing the cellular components comprises exposing the biological sample to an enzyme able to degrade sugars or sugar-modified biomolecules.

204. The method of any one of claims 1-192, wherein clearing the cellular components comprises exposing the biological sample to a detergent.

205. The method of any one of claims 1-192, wherein clearing the cellular components comprises exposing the gel to a proteinase.

206. The method of any one of claims 1-192, wherein clearing the cellular components comprises exposing the polymer matrix to guanidine HCl207. The method of any one of claims 1-192, wherein clearing the cellular components comprises exposing the gel to Triton X-100 (polyethylene glycol p- (1,1, 3, 3-tetramethylbutyl) -phenyl ether).

208. The method of any one of claims 1-192, wherein clearing the cellular components comprises exposing the polymer matrix to sodium dodecyl sulfate.

209. The method of any one of claims 1-192, wherein clearing the cellular components comprises exposing the polymer matrix to ethylenediaminetetraacetic acid.

210. The method of any one of claims 53-101, wherein the imaging comprises using epi-fluorescence microscopy, total-internal-reflectance microscopy, highly inclined thin-illumination (HILO) microscopy, light-sheet microscopy, scanning confocal microscopy, scanning line confocal microscopy, spinning disk confocal microscopy, or other comparable conventional microscopy techniques.

211. A bridge nucleic acid probe comprising: an acrydite moiety and a target sequence configured to hybridize with a distinct nucleic acid species in a biological sample.

212. A plurality of bridge nucleic acid probe pools comprising bridge probes, the bridge probes comprising: a target sequence and an acrydite moiety, wherein each bridge nucleic acid probe pool is configured to hybridize to a distinct RNA species in a sample.VIZG019.PCT / US213. The bridge nucleic acid probe pools of claim 212, wherein the bridge probes comprise a target sequence, with an average length of between 10 and 200 nucleotides, configured to hybridize the distinct RNA species.

214. The bridge nucleic acid probe pools of claim 212, wherein each bridge nucleic acid probe pool comprises at least 10 different target sequences.

215. The bridge nucleic acid probe pools of claim 212, wherein the bridge nucleic acid probes arc covalently bonded to an anchoring agent.

216. The bridge nucleic acid probe pools of claim 215, wherein the anchoring agent is an alkylating agent.

217. The bridge nucleic acid probe pools of claim 216, wherein the alkylating agent is selected from the group consisting of Altretamine, Bendamustine, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine, Ifosfamide, Lomustine, Mechlorethamine, Melphalan, Oxaliplatin, Temozolomide, Thiotepa and Trabectedin.

218. The bridge nucleic acid probe pools of claim 215, wherein the anchoring agent and the bridge nucleic acid probes each comprise an acrydite moiety configured to covalently bind a polymer matrix.

219. The bridge nucleic acid probe pools of claim 215, wherein the anchoring agent is an alkylating agent denvatized with an acrydite moiety.

220. The bridge nucleic acid probe pools of claim 215, wherein the bridge nucleic acid probes are embedded in a polymer matrix and the anchoring agent and acrydite moiety of the bridge nucleic acid probe are each covalently bonded the polymer matrix.

221. The bridge nucleic acid probe pools of claim 220, wherein the polymer matrix is a polyacrylamide gel.

222. The bridge nucleic acid probe pools of claim 220, wherein the bridge nucleic acid probe pools are hybridized to a plurality of primary nucleic acid probe pools each of the primary nucleic acid probes comprising a bridge probe target sequence and one or more read sequences, wherein each pool of primary nucleic acid probes hybridize to a distinct bridge nucleic acid probe pool and each pool of primary probes encode a N-bit code that was assigned to each distinct RNA species.VIZG019.PCT / US223. The bridge nucleic acid probe pools of claim 222, wherein each assigned N-bit code is a valid codeword.

224. The bridge nucleic acid probe pools of claim 223, wherein the valid codewords have a Hamming distance of 2 between valid codewords.

225. The bridge nucleic acid probe pools of claim 222, wherein each of the read sequences in a primary probe pool correspond to a bit value of 1 for the codewords assigned to the distinct RNA species.

226. The bridge nucleic acid probe pools of claim 223, wherein the valid codewords have a Hamming distance equal to or greater than 4 between each of the valid codewords.

227. The bridge nucleic acid probe pools of claim 222, wherein each primary nucleic acid probe pool comprises at least 10 different primary nucleic acid probes.

228. The bridge nucleic acid probe pools of claim 222, wherein each primary nucleic acid probe pool comprises at least four distinct read sequences.

229. The bridge nucleic acid probe pools of claim 222, wherein each primary nucleic acid probe pool comprises four distinct read sequences.

230. The bridge nucleic acid probe pools of claim 222, wherein each primary nucleic acid probe pool comprises at least eight distinct read sequences.

231. The bridge nucleic acid probe pools of claim 157, wherein the primary nucleic acid probes comprise a target sequence, with an average length of between 10 and 200 nucleotides, configured to hybridize the distinct bridge nucleic acid probe pools.

232. The bridge nucleic acid probe pools of claim 222, wherein each primary nucleic acid probe pool comprises at least 10 different target sequences.The bridge nucleic acid probe pools of claim 222, wherein the N-bit code comprises at least a 16-bit code.

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