Methods for anchoring fragmented nucleic acid targets to polymer matrices for imaging - Patent Application 20070123333

Abstract

The present disclosure generally relates to a method of anchoring nucleic acid to a matrix and then imaging nucleic acid targets (e.g. RNA transcript molecules) in tissue samples, such as formalin-fixed paraffin-embedded (FFPE) tissue sections, where the nucleic acid may be fragmented. For example, a method of anchoring target nucleic acid in a matrix and removing non-target cellular components is provided, the method comprising: contacting a tissue sample with at least two anchoring agents, the first anchoring agent forming a covalent bond with the target nucleic acid and the second anchoring agent comprising an oligonucleotide that hybridizes with the target nucleic acid; embedding the sample in a polymer matrix, the first and second anchoring agents each forming a covalent bond with the polymer matrix; and removing the non-target cellular components from the polymer matrix, leaving the target nucleic acid anchored in the polymer matrix to form a matrix-anchored target nucleic acid sample.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 311,319, filed February 17, 2022; and U.S. Patent Application No. 63 / 424,891, filed November 12, 2022, each of which is incorporated herein in its entirety.

[0002] Technical Field The present application relates generally to the field of in situ imaging, and more particularly to methods for determining nucleic acid targets within tissue samples. [Background technology]

[0003] To accurately profile gene expression in tissue samples in situ, spatial transcriptome techniques with high detection efficiency and single molecule resolution are required. In situ single-cell transcriptome imaging techniques, such as multiplexed error-robust fluorescence in situ hybridization (MERFISH), allow for direct profiling of the spatial organization of intact tissues with subcellular resolution.

[0004] However, background from factors such as cellular autofluorescence often makes in situ imaging applications in tissue samples more challenging. Without wishing to be bound by any theory, it is believed that certain components, such as proteins and lipids, unbound or unrelated nucleic acids, fluorescent components (bleached or unbleached), can cause problems in imaging or analysis, for example, due to autofluorescence, components that quench fluorescent molecules, off-target binding, or other phenomena. For example, it is believed that a nucleic acid probe may not bind to the appropriate target in the sample, but instead bind "off-target" to other cellular components, including but not limited to proteins, lipids, RNA, DNA, etc. Similarly, a probe targeted to one DNA or RNA molecule may bind "off-target" to the wrong DNA or RNA molecule. These interactions may be driven, for example, by imperfect base pairing, charge-charge interactions, or other molecular interactions. Thus, a polymer matrix or gel can be applied to the sample to immobilize the desired nucleic acid molecule (or other desired target) while removing or degrading components to which the nucleic acid probe binds off-target ("non-target cellular components") from the sample. This can reduce the amount of probes that bind off-target, facilitating imaging or other analysis of the sample. Other components, such as proteins and lipids, can be removed or degraded from the sample. This can reduce the amount of background, facilitating imaging or other analysis of the sample. The methodology developed to remove cellular components from gel-embedded tissue sections works well enough with fresh and fixed frozen samples, where anchor oligonucleotides and target probes are added to the sample, then embedded in gel, and cellular components are removed or removed before imaging ("Protocol B"). See FIG. 1. The method provided herein is insufficient for fixation of target nucleic acid for imaging and hybridization of target probe to nucleic acid when used with formalin-fixed paraffin-embedded ("FFPE") tissue sections.

[0005] For example, a method for preparing a sample for MERFISH imaging is disclosed in U.S. Patent Publication No. 2019 / 0264270, entitled "Matrix Imprinting and Clearing," the contents of which are incorporated herein by reference. MERFISH imaging has been previously demonstrated in fresh frozen tissue blocks and fixed frozen tissue blocks, but not in formalin-fixed paraffin-embedded (FFPE) tissue sections. In fact, FFPE tissue sections are the most widely used type of clinical sample in histology and molecular diagnostics, but FFPE samples are known to be often incompatible with in situ single-cell transcriptome analysis due to RNA degradation and protein cross-linking. Thus, there is a need for methods and / or reagents that enable in situ single-cell transcriptome analysis from FFPE tissue samples. Thus, the present disclosure provides improved methods of imaging nucleic acid targets, including preparation of tissue samples that enable in situ single-cell transcriptome imaging (e.g., MERFISH) from FFPE tissue sections. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] US Patent Publication No. 2019 / 0264270 Summary of the Invention

[0007] Described herein is a method and its reagent for in situ single-cell transcriptome analysis from FFPE tissue sections or other tissue samples suspected of nucleic acid fragmentation.In some embodiments, provided herein is a method for anchoring and imaging target nucleic acid molecules (e.g., mRNA transcripts) from FFPE tissue samples, the method comprising: a) contacting the tissue sample with at least two anchoring agents, a first anchoring agent that forms a covalent bond with the target nucleic acid, and a second anchoring agent that comprises an oligonucleotide that hybridizes with the target nucleic acid; b) embedding the sample in a polymer matrix, in which the first and second anchoring agents each form a covalent bond with a polymer gel; c) removing non-target cellular components from the polymer matrix, and the target nucleic acid remains anchored in the polymer matrix to form a matrix-anchored target nucleic acid sample.In certain embodiments, the method further comprises an imaging step d) of contacting the anchored target nucleic acid sample with one or more primary oligonucleotide probes that hybridize with the target nucleic acid, and a plurality of secondary nucleic acid probes that comprise a fluorescent label and a recognition sequence that hybridize to the sequence of the primary nucleic acid probe, and imaging the target nucleic acid. [Brief description of the drawings]

[0008] [Figure 1] Figure 1 shows a comparison of Protocol A (the method of the present disclosure) and Protocol B (comparative protocol). See Example 1.

[0009] [Diagram 2]Figures 2A-2C show MERFISH imaging using a 128-plex gene panel in FFPE mouse small intestine tissue sections according to Example 1 (Protocol A). Figure 2A shows the spatial distribution of selected genes throughout the tissue; Figure 2B shows tissue morphology visualized by selected transcripts (left) and the distribution of all transcripts in a magnified area; Figure 2C shows the correlation of MERFISH counts with bulk RNA sequencing FPKM (fragments per kilobases per million) data, demonstrating that the measurements are quantitative and highly accurate. Scale bar: 1 mm. See Example 2.

[0010] [Diagram 3] Figures 3A-3C show MERFISH imaging using a 244-plex gene panel in FFPE human colon cancer tissue sections according to Example 1 (Protocol A). Figure 3A: Spatial distribution of selected genes across tissue; Figure 3B: Tissue morphology visualized by selected transcripts (left) and distribution of all transcripts in a zoomed-in area; Figure 3C: Correlation of MERFISH counts with bulk RNA sequencing FPKM data. Scale bar: 1 mm. See Example 2.

[0011] [Figure 4] Figures 4A-4C show MERFISH imaging using a 483-plex gene panel in FFPE mouse brain tissue sections according to Example 1 (Protocol A). Figure 4A: Spatial distribution of selected genes across the tissue; Figure 4B: Tissue morphology visualized by selected transcripts (left) and distribution of all transcripts in a magnified area; Figure 4C: Correlation of MERFISH counts with bulk RNA sequencing data. Scale bar: 1 mm. See Example 2.

[0012] [Diagram 5]FIG. 5 shows a comparison of sample preparation according to the protocol of Example 1 ("Protocol A") with a comparative protocol ("Protocol B"). For Protocol B, MERFISH probes (e.g., primary oligonucleotide probes) and anchor probes are added before the tissue removal step. FFPE mouse small intestine samples were processed with Protocol A or Protocol B, and fresh frozen mouse small intestine samples were processed with Protocol B. The average transcript counts per field of view (FOV) for both conditions are shown. N=3 See Example 3 and FIG. 1.

[0013] [Figure 6A] Figure 6A-C show MERFISH imaging using a 244-plex gene panel in 15 different archival human FFPE tissue section samples. For each dataset, 1000-2000 fields of view were captured, generating millions to hundreds of millions of counts per tissue slice. Figure 6A shows the average MERFISH counts per field of view at an area size of 200 x 200 µm, demonstrating that the workflow works robustly across a wide range of human normal and cancer FFPE tissue section samples. [Figure 6B] Figure 6B shows that the quality of the MERFISH data correlates with the quality of the RNA in the sample, as shown by DV200 values ​​of greater than 40%. DV200 is the percent of RNA fragments greater than 200 nucleotides in a sample. [Figure 6C] Figure 6C shows the correlation of MERFISH counts across different tissues with bulk RNA sequencing FPKM data. See Example 4.

[0014] [Figure 7] Figure 7 shows a comparison of samples prepared according to the protocol of Example 1 ("Protocol A") with a comparative method ("Protocol B") across different sample types, showing the average counts per field of view with an area size of 200 x 200 μm across different tissue types, and the correlation of MERFISH data with the correlation coefficients included for each tissue type. See Example 5.

[0015] [Figure 8] Figure 8 shows that protocol A outperforms protocol B in a variety of fresh frozen (FF) human samples while maintaining precision of the measurements. Figure 8A) Fresh frozen human lymph node, lung, colon and kidney samples were processed with a panel of 244 genes with protocol A (first anchoring the target nucleic acid and hybridizing after polymer matrix embedding and removal of non-target cellular components) or protocol B. B, and all samples were then imaged with MERSCOPE. The average counts of transcripts per field of view (FOV) for both conditions are shown.

[0016] [Figure 9] Figure 9 shows that protocol A outperforms protocol B in a range of fresh frozen (FF) human samples while maintaining precision of measurement. Transcripts imaged per gene with protocol A correlated with protocol B in human lung and kidney samples. The correlation coefficients were 0.99 and 0.98, respectively, indicating that protocol A was able to reproduce expression levels measured by protocol B, and overall there were substantially more counts with protocol A.

[0017] [Figure 10]Figures 10A-C show that the FFPE workflow is highly sensitive, accurate and reproducible. Figure 10A shows the correlation of MERSCOPE data between two human ovarian cancer slices from the same patient. The correlation coefficient is 0.99, indicating high reproducibility of the measurements. Figure 10B shows a human ovarian cancer sample 1 analyzed by MERSCOPE using a 500-gene panel and an adjacent slice analyzed by bulk RNA sequencing. The correlation analysis between MERFISH counts and FPKM values ​​from bulk RNA sequencing is shown. The correlation coefficient is 0.82, indicating high precision of the measurements. Figure 10C presents the correlation analysis between MERSCOPE data and bulk RNA sequencing performed across 14 cancer samples, where the correlation coefficient indicates high precision across multiple cancer types and replicates. See Example 8.

[0018] [Figure 11] Figures 11A-11F show that the FFPE cell segmentation workflow enables true atlasing in dense tissues. Figure 11A shows FFPE human liver cancer immunostained with the Cell boundary staining kit and DAPI for nuclear staining. Figure 11B shows cells segmented using a deep learning-based cell segmentation algorithm. Polygon masks of each identified cell are shown. Figure 11C shows a UMAP visualization of the 17 different cell types identified in human liver cancer, generated from MERFISH transcript data. Figure 11D shows the spatial distribution of cell types across the tissue within the boxed region of Figure 11B. Figure 11E shows the spatial distribution of fibroblasts in the boxed region of Figure 11B. Fibroblast marker gene COL1A1 (shown in yellow). Figure 11F shows the local distribution of endothelial cells in the boxed area in B. The endothelial marker gene PECAM1 is shown in green. See Example 8.

[0019] [Figure 12] Figure 12 shows the spatial distribution of identified cell types across different FFPE tumor samples. Different cancer samples, including breast, colon, melanoma, lung, liver, ovarian, prostate and uterine cancer, were analyzed by MERSCOPE using a 500 gene panel with cell border staining kit to label cell borders. Cells are segmented and subjected to single cell analysis. Identified cells in each sample are colored to show the spatial distribution of different cells across the sample. Scale bar: 1 mm. See Example 8.

[0020] [Figure 13] FIG. 13 shows that the FFPE protocol can be used to show the spatial distribution of expression of select genes (ACTA2, CD3D, LGR5, MK167 and PECAM1) in human breast cancer. FIG. 13A shows the spatial distribution of select genes including ACTA2 (green), CD3D (red), LGR5 (light green), MKI67 (magenta) and PECAM1 (blue) from 500 genes analyzed across the tissue. Scale bar: 1 mm. FIG. 13B provides a magnification of the boxed area in FIG. 13A. Scale bar: 1 mm. FIG. 13C shows a magnification of the boxed area in FIG. 13B, showing the cell boundary polygon mask in grey. Scale bar: 250 mm. See Example 8.

[0021] [Figure 14A] Figures 14A-E show that FFPE protocols can be used to identify and map cell types in human breast cancer. Figure 14A provides a UMAP visualization of the different cell types identified in human breast cancer, generated from MERFISH transcript data. [Figure 14B-D]Figure 14B shows the spatial distribution of the 14 identified cell types across the tissue. Figure 14C shows the spatial distribution of the identified cell types within the boxed area of ​​Figure 14B. Figure 14D shows the spatial distribution of two types of fibroblasts (Fibroblast 1 in green and Fibroblast 2 in red) within the boxed area of ​​Figure 14C. Both types of fibroblasts express the COL1A1 gene, while Fibroblast 2 expresses the proliferation marker MKI67. [Figure 14E] Figure 14E provides a dot plot showing the marker genes for each cell type. See Example 8.

[0022] [Figure 15A-B] Figure 15 shows that FFPE protocols can be used to characterize immune cell types in the tumor microenvironment. Figure 15A shows that T / NK cell clusters from breast cancer samples were selected for subclustering analysis. UMAP visualization of the subclustering analysis shows seven distinct immune cell subtypes within human breast cancer. Figure 15B provides dot plots showing marker genes for each immune cell type, including myeloid cells, CD4+ T cells, CD8+ T cells, CD4+ regulatory T cells (Treg), and NK lineage cells. [Fig. 15C-E] Figure 15C provides the spatial distribution of Tregs. Figure 15D provides the spatial distribution of CD4+ T cells. Figure 15E provides the spatial distribution of selected genes in the expanded region in human breast cancer, showing CD4, CD8A, FOXP3, NCR1 and CTLA4. Note that FOXP3 positive Tregs express T cell exhaustion markers. See Example 8. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present disclosure generally relates to the preparation of tissue samples for in situ imaging when nucleic acid, particularly mRNA, fragmentation is suspected. In an embodiment, the sample is a FFPE tissue sample. The present disclosure also provides for the preparation of tissue samples that allow in situ single-cell transcriptome imaging (e.g., MERFISH, smFISH) to detect nucleic acid targets in the sample. The method of the present disclosure can be used to prepare gene expression profiles of tissue samples. Other aspects generally relate to systems or kits that include such methods.

[0024] In certain embodiments, the present specification provides methods and compositions for FFPE sample preparation and its use in transcriptome analysis.In some embodiments, transcriptome analysis includes single molecule (sm) FISH, barcoding (also referred to herein as "codeword") method for quantifying transcriptome-wide, and combinatorial barcoding (e.g., seqFISH) for quantitative spatial transcriptome analysis, or error correction method including hybridization chain reaction (HCR) seqFISH and multiplex error-robust (MER) FISH.

[0025] In certain embodiments provided herein, a method is provided for anchoring target nucleic acid in a matrix and removing non-target cellular components, which can then be used for downstream imaging of nucleic acid target.In some embodiments, the method includes contacting a tissue sample suspected of containing fragmented nucleic acid (e.g., formalin-fixed paraffin-embedded (FFPE) sample) with at least two anchoring agents, a first anchoring agent that forms a covalent bond with the target nucleic acid, and a second anchoring agent that comprises an oligonucleotide that hybridizes with the target nucleic acid; embedding the sample in a polymer matrix, in which the first and second anchoring agents each form a covalent bond with the polymer matrix; and removing the non-target cellular components from the polymer matrix, leaving the target nucleic acid anchored in the polymer matrix to form a matrix-anchored target nucleic acid sample. In certain embodiments, the target nucleic acid is RNA, particularly mRNA, and the method includes contacting a tissue sample suspected of containing fragmented RNA (e.g., a formalin-fixed, paraffin-embedded (FFPE) tissue sample) with at least two anchoring agents, a first anchoring agent comprising an alkylating agent that forms a covalent bond with the target nucleic acid, and a second anchoring agent comprising a poly-T sequence that is complementary to and hybridizes with the target RNA, wherein the first anchoring agent and the second anchoring agent each comprise an acrydite moiety that is covalently attached to the matrix; embedding the sample in a polymer matrix, wherein the first and second anchoring agents each form a covalent bond with the polymer matrix; and removing non-target cellular components from the polymer matrix, leaving the target RNA anchored to the polymer matrix to form a matrix-anchored target RNA sample.

[0026] In certain embodiments, FFPE tissue sections are prepared for transcriptome analysis (e.g., RNA transcripts) including the steps of deparaffinization, ethanol rehydration, antigen retrieval, followed by the addition of at least two anchoring agents (e.g., two functionally distinct or separate anchoring agents) to the tissue sample, where 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 is functionalized with two separate anchoring agents to form an anchored tissue sample. This anchoring step improves the immobilization of the target nucleic acid within a gel or polymer matrix, particularly mRNA, which may be fragmented during the FFPE process of fixing the tissue section. Each of these anchoring agents further comprises a chemical moiety (e.g., a reactive group) that can form a covalent bond with the polymer matrix either while the polymer matrix is ​​being formed (during polymerization) or after. Thus, after at least two anchoring agents are added to tissue sample, the sample is embedded in a polymer matrix, and the first and second anchoring agents each form a covalent bond with the polymer matrix.In this way, target nucleic acid is 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 (e.g., acryldite) that forms a covalent bond with acrylamide.

[0027] After the gel embedding step, non-target 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). This clearing step, in combination with the use of at least two anchoring agents when starting with an FFPE tissue sample, removes protein crosslinks induced by the formalin fixation process, exposing the target nucleic acid to a complementary primary oligonucleotide probe designed to hybridize to a target sequence in the anchored nucleic acid. Comparative protocol B disclosed herein (see FIG. 1) includes a step of adding a primary oligonucleotide probe before the clearing step, which is sufficient for fresh fixed frozen samples, but significantly reduces the number of transcripts imaged with FFPE samples. See FIG. 4. Applicants have found that the use of a combination of two anchoring agents, as well as the order of steps performed with, for example, a primary oligonucleotide probe added after anchoring and clearing, significantly improves the target nucleic acid available for hybridization and visualization.

[0028] Thus, after the final step of removing tissue, the original FFPE tissue sample is prepared and ready for transcriptome analysis according to protocols known to those skilled in the art (e.g., probe hybridization and imaging).In certain embodiments, a method of imaging target nucleic acid in a matrix and removing non-target cellular components is provided herein, the method comprising: contacting a formalin-fixed paraffin-embedded (FFPE) tissue sample, or a sample suspected of containing fragmented nucleic acid, with at least two anchoring agents, the first anchoring agent forming a covalent bond with the target nucleic acid, and the second anchoring agent comprising an oligonucleotide hybridizing with the target nucleic acid; embedding the sample in a polymer matrix, the first and second anchoring agents each forming a covalent bond with the polymer matrix; removing the non-target cellular components from the polymer matrix, leaving the target nucleic acid anchored in the polymer matrix to form a matrix-anchored target nucleic acid sample; and contacting the anchored target nucleic acid sample with one or more primary oligonucleotide probes hybridizing to the target nucleic acid, and a plurality of secondary nucleic acid probes comprising a fluorescent label and a recognition sequence hybridizing to the sequence of the primary nucleic acid probe, and imaging the target nucleic acid.

[0029] In certain embodiments, the method includes contacting the nucleic acid target (e.g., an RNA transcript) with at least two anchoring agents to improve the efficiency of immobilization of the RNA transcript prior to polymer matrix embedding. In embodiments, the method includes removing tissue (e.g., removing non-target cellular components) prior to contacting the sample with a primary oligonucleotide probe (e.g., a MERFISH probe, a smFISH probe, etc.) to increase the efficiency of primary probe binding by exposing the target nucleic acid after cross-linking proteins have been removed. In certain embodiments, the method includes contacting an RNA transcript (e.g., a nucleic acid target) with at least two anchoring agents, where a first anchoring agent forms a covalent bond with the target nucleic acid and a second anchoring agent (anchor probe) comprises an oligonucleotide that hybridizes to the target nucleic acid, embedding a tissue sample comprising the at least two anchoring agents in a gel matrix such that the RNA transcript is immobilized in a polymer gel matrix when the first and second anchoring agents each form a covalent bond with the polymer matrix, digesting or removing tissue and / or non-target cellular components, thereafter contacting the immobilized nucleic acid (e.g., RNA transcript) with a plurality of primary oligonucleotide probes that specifically hybridize to the immobilized target nucleic acids, and imaging the target nucleic acids.

[0030] In certain embodiments provided herein, kits for FFPE sample preparation are provided that include one or more of the following reagents: deparaffinization buffer, decrosslinking buffer, conditioning buffer, sample preparation wash buffer, formamide wash buffer, gel embedding premix, removal premix, gel coverslip, pre-anchor activator, anchor buffer, and digestion premix. In certain embodiments, the kits of the present disclosure include at least two "anchor buffer" formulations, one containing a first anchor agent and the other containing a second anchor agent or anchor probe.

[0031] An exemplary method and its kit components according to the present disclosure are described in Example 1.

[0032] Thus, in one set of embodiments, a method is provided for detecting (e.g., by imaging) a nucleic acid target in a tissue sample (e.g., FFPE), the method including contacting a sample containing a nucleic acid target with an anchor probe that specifically binds to the nucleic acid target (e.g., via hybridization); immobilizing the nucleic acid target-binding anchor probe to at least a portion of the sample within a gel (e.g., via covalent attachment of the anchor probe to a polymer gel); removing the tissue sample within the polymer gel by removing or degrading non-target cellular components (e.g., non-immobilized target nucleic acids); contacting the sample with a plurality of primary nucleic acid probes that can selectively bind (e.g., hybridize to) the nucleic acid target; and detecting (e.g., by imaging) the nucleic acid probes bound to the nucleic acid target in the removed (e.g., gel-embedded) sample.

[0033] In some embodiments, a method of detecting (e.g., by imaging) a nucleic acid target in a tissue sample (e.g., FFPE) is provided, the method comprising: contacting a tissue sample containing a nucleic acid target with an anchoring agent comprising a first chemical moiety (e.g., a reactive group capable of forming a covalent bond with the target nucleic acid) capable of reacting with and / or modifying an internal base of the nucleic acid target, and a second chemical moiety (e.g., a reactive group capable of forming a covalent bond with the polymer gel) that can be incorporated into a polymer gel; immobilizing the nucleic acid target-binding anchoring agent to at least a portion of the sample within the gel (e.g., via covalent attachment of the anchoring agent to the polymer gel); removing the tissue sample within the polymer gel by removing or degrading non-targets (e.g., non-immobilized target nucleic acids); contacting the tissue sample with a plurality of primary nucleic acid probes capable of selectively binding (e.g., hybridizing) to the nucleic acid targets; and detecting (e.g., by imaging) the nucleic acid probes bound to the nucleic acid targets within the removed tissue (e.g., gel-embedded) sample.

[0034] In some embodiments, a method of detecting (e.g., by imaging) a nucleic acid target in a tissue sample (e.g., FFPE) is provided, the method comprising: contacting a tissue sample containing a nucleic acid target with an anchoring agent that includes a first chemical moiety that can react with and / or modify an internal base of the nucleic acid target (e.g., a reactive group that can form a covalent bond with the target nucleic acid) and a second chemical moiety that can be incorporated into a polymer gel (e.g., a reactive group that can form a covalent bond with a polymer gel); contacting the tissue sample with an anchor probe that specifically binds (e.g., by hybridization) the nucleic acid target; The method includes immobilizing a selectively-binding anchor probe and a nucleic acid target-binding anchor agent to at least a portion of the sample within the gel (e.g., via covalent attachment of the anchor agent to the polymer gel); removing the tissue sample within the polymer gel by removing or degrading non-targets (e.g., non-immobilized target nucleic acids); contacting the removed tissue sample (e.g., immobilized target nucleic acid sample) with a plurality of primary oligonucleotide nucleic acid probes capable of selectively binding (e.g., hybridizing) to the nucleic acid targets; and detecting (e.g., by imaging) the nucleic acid probes bound to the immobilized nucleic acid targets within the removed (e.g., gel-embedded) sample.

[0035] In certain embodiments, a method for imaging target nucleic acid from a formalin-fixed paraffin-embedded (FFPE) tissue sample is provided.In some embodiments, the method includes: contacting the tissue sample with at least two anchoring agents, 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; embedding the sample in a polymer gel, the first and second anchoring agents each form a covalent bond with the polymer gel; removing non-immobilized cellular components from the polymer gel to form a gel-immobilized target nucleic acid sample; and contacting the immobilized target nucleic acid with one or more primary oligonucleotide probes that hybridize with the target nucleic acid, and imaging the target nucleic acid sample.

[0036] In some embodiments, the step of removing is performed after the target nucleic acid is immobilized in the polymer gel (e.g., after the steps of contacting the sample with anchor probes and anchoring agents and immobilizing the nucleic acid target binding anchor probes and nucleic acid target binding anchoring agents) and before adding the primary oligonucleotide probes to the immobilized target nucleic acid. For example, in certain embodiments, the desired target is immobilized within a gel (such as an inert gel matrix) and other components are removed or degraded.

[0037] The primary oligonucleotide probes may be, for example, MERFISH or smFISH probes, and may be substantially complementary to mRNA or other RNA, for example, for transcriptome analysis. The primary oligonucleotide probes may also include a signaling entity, for example, a fluorescent signaling entity, for imaging and / or analysis of the sample. In certain embodiments, the secondary oligonucleotide probes hybridized to the primary oligonucleotide probes include an imaging moiety (e.g., a fluorescent signaling entity), and imaging includes adding one or more secondary probes. In some embodiments, the method further includes creating a codeword or barcode based on the distribution of bound nucleic acid probes in the sample. In some embodiments, the method further includes, for at least some of the codewords, matching the codeword with a valid codeword, and if no match is found, optionally applying error correction to the codeword to form a valid codeword or discarding the codeword. I. Tissue Samples

[0038] As used herein, a "tissue sample" herein refers to a collection of similar cells obtained from a subject's tissue. The tissue may contain nucleated cells with chromosomal material. The source of the tissue sample may be solid tissue from a fresh, frozen, FFPE, and / or preserved organ or tissue sample, or a biopsy, or an aspirate, or blood, or any blood component, or a bodily fluid, such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid, or cells from any time point in the subject's pregnancy or development. The tissue sample may also be primary cells or cultured cells or cell lines, or cultured tissue. The tissue sample may contain compounds that are not naturally mixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc. In some embodiments of the present invention, the tissue sample is a non-hematological tissue (i.e., not blood or bone marrow tissue). In some embodiments, the tissue sample used in the method is a formalin-fixed paraffin-embedded tissue sample. Although the method can be used with any of the tissue samples disclosed herein, the method provides particular advantages for FFPE tissue samples, or any sample suspected of containing fragmented nucleic acid and / or accessible nucleic acid targets.

[0039] In some embodiments, nucleic acid fragmentation can be assessed and determined using methods well known in the art. For example, to assess the quality of a sample for in situ hybridization, it is beneficial to determine the quality of the RNA of the tissue block using the RNA Integrity Number (RIN) or DV200 value via commercially available instruments such as the BioAnalyzer or TapeStation platform. Briefly, RNA from the sample is first extracted and measured on either the BioAnalyzer or TapeStation. RIN is expressed as a value ranging from 1 to 10, with 1 indicating that the sample has shorter, more highly degraded RNA and 10 reflecting longer, less degraded RNA. A higher RIN score will have more intact 18S and 28S RNA. DV200 reflects the percentage of RNA fragments in the tissue sample that are greater than 200 nucleotides in length. Tissues with a lower DV200 percentage have shorter, more degraded RNA. Conversely, a higher percentage indicates that longer, less degraded RNA molecules are present in the tissue.

[0040] In some embodiments, the tissue sample is a tissue section, a clinical smear, or cultured cells or tissue. In some embodiments, the tissue sample comprises a tissue section. As used herein, a "section" of a tissue sample herein refers to a single portion or piece of a tissue sample, e.g., a thin slice of tissue or cells cut from a tissue sample. It is understood that multiple sections of a tissue sample can be taken and subjected to analysis according to the present invention. In some embodiments, the selected portion or section of the tissue comprises a homogenous population of cells. In some embodiments, the selected portion or section of the tissue comprises a heterogenous population of cells. In some embodiments, the selected portion comprises an area of ​​the tissue, e.g., the lumen, as a non-limiting example. The selected portion may be as small as, e.g., one or two cells, or may represent thousands of cells.

[0041] Any tissue sample from a subject can be used. Examples of tissue samples that can be used include, but are not limited to, breast, prostate, ovary, colon, lung, endometrium, stomach, salivary gland or pancreas. Tissue samples can be obtained by a variety of procedures, including, but not limited to, surgical resection, aspiration or biopsy. Tissues can be fresh or frozen.

[0042] In some embodiments, the tissue section is a mammalian (e.g., human or mouse) tissue section of brain, adrenal gland, colon, small intestine, stomach, heart, liver, skin, kidney, lung, pancreas, testis, ovary, prostate, uterus, thyroid, and spleen. 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 a mouse small intestine. In some embodiments, the tissue sample is from a mouse brain. In some embodiments, the tissue sample is from a human liver cancer. In some embodiments, the tissue sample is from a human kidney. In some embodiments, the tissue sample is from a human lung. In some embodiments, the tissue sample is from a human ovarian cancer. In some embodiments, the tissue sample is from a human uterine cancer. In some embodiments, the tissue sample is from a human lung cancer.

[0043] In some embodiments, the tissue is stored for a period of time, for example, frozen or FFPE.In some embodiments, the tissue sample is a frozen tissue sample.In some embodiments, the tissue is a frozen tissue.In some embodiments, the tissue is a paraffin-embedded tissue.In some embodiments, the tissue is a formalin-fixed paraffin-embedded tissue. A. Tissue Sample Preparation 1. Tissue Sample Acquisition and Fixation

[0044] Tissue samples can be obtained from intact organs or tissues using any method well known to those of skill in the art, such as conventional methods used to prepare tissue samples for immunohistochemistry (IHC) or in situ hybridization (ISH) techniques.

[0045] For example, any intact organ or tissue can be cut into appropriately small pieces (one or more) (the size of the cut pieces typically ranges from a few millimeters to a few centimeters) and "fixed" to preserve the location of the nucleic acids within the sample. Techniques for fixing cells and tissues are known to those of skill in the art. Non-limiting examples of fixatives include formaldehyde, paraformaldehyde, glutaraldehyde, ethanol, methanol, acetone, acetic acid, and the like.

[0046] 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 some embodiments, the tissue sample is formalin fixed paraffin embedded (FFPE).

[0047] In addition to intact samples, other samples may be used. In some embodiments, frozen sections are prepared by rehydrating 50 mg of frozen ground tissue at room temperature in phosphate buffered saline (PBS) in a small plastic capsule, pelleting the particles by centrifugation, resuspending the particles in viscous embedding medium (OCT), inverting the capsule and / or pelleting again by centrifugation, flash freezing in isopentane at -70°C, cutting the plastic capsule and / or removing the frozen cylinder of tissue, fixing the tissue cylinder on a cryostat microtome chuck, and Similarly, permanent tissue sections may be prepared by rehydrating a 50 mg sample in a plastic microcentrifuge tube, pelleting, resuspending in 10% formalin for 4 hours; washing / pelleting; resuspending in warm 2.5% agar, pelleting, chilling 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 consecutive permanent sections.

[0048] In some embodiments, the present invention can utilize standard frozen samples, such as those embedded in OCT and not milled, including those used in standard frozen section hospital laboratories.

[0049] Tissue samples are often fixed by conventional methodology. Formaldehyde 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 Kopinski, J., Histochem.Cytochem., 34:1095 (1986). Those skilled in the art will understand that the choice of fixative is determined by the purpose for which the tissue is histologically stained or otherwise analyzed. Those skilled in the art will also understand that the length of fixation depends on the size of the tissue sample and the fixative used.

[0050] The sample used may 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 formalin-fixed paraffin embedded. In some embodiments, the formalin-fixed paraffin-embedded (FFPE) tissue block is hematoxylin and eosin (H&E) stained. As is commonly known in the art, the tissue sample may be first fixed, then dehydrated through a series of ascending alcohols, infiltrated and embedded in paraffin or other sectioning medium so that the tissue sample can be sectioned. Alternatively, the tissue may be sectioned and the resulting sections fixed. By way of example, the tissue sample may be embedded in paraffin and processed by conventional methodology. Examples of paraffins that may be used include, but are not limited to, Paraplast, Broloid, and Tissuemay. After embedding the tissue sample, it may be sectioned, such as with a microtome. 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, etc. By way of example, paraffin-embedded sections can be attached to positively charged slides and / or slides coated with poly-L-lysine.

[0051] In some embodiments, the tissue sections may be in the range of about 3 μm to about 100 μm, or any intermediate range therein. In some embodiments, the tissue sections may be in the range of about 10 μm to about 100 μm. In some embodiments, the tissue sections may be in the range of about 10 μm to about 50 μm. In some embodiments, the tissue sections may be in the range of about 10 μm to about 30 μm. In some embodiments, the tissue sections may be in the range of about 10 μm to about 15 μm. In some embodiments, the tissue sections may be in the range of about 3 μm to about 15 μm. In some embodiments, the tissue sections may be in the range of about 5 μm to about 20 μm. In some embodiments, the tissue sections may be in the range of about 15 μm to about 30 μm. In some embodiments, the tissue sections may be in the range of about 3 μm, about 4 μm, about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, about 10 μm, about 11 μm, about 12 μm, about 13 μm, about 14 μm, about 15 μm, or about 20 μm. In some embodiments, the tissue sections may be in the range of about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, or about 100 μm. 2. Deparaffinization and Rehydration

[0052] Tissue sections can be deparaffinized using methods known in the art and / or commercially available kits. This method removes the bulk of the paraffin from the sample. Various techniques for deparaffinization are known, including, but not limited to, washing with organic solvents or agents to dissolve the paraffin.

[0053] Exemplary deparaffinizing solvents include, but are not limited to, benzene, toluene, ethylbenzene, xylene, D-limonene, octane, and mixtures thereof. In certain embodiments, the deparaffinizing solvent comprises D-limonene. These solvents are preferably of high purity, usually greater than 99%. The volume used and number of washes required depends on the size of the sample and the amount of paraffin to be removed. Samples may be washed from 1 to about 10 times, or from about 2 to about 4 times. A typical volume of organic solvent is about 500 ml for a 10 mm tissue sample.

[0054] After deparaffinization, the sample may be rehydrated, for example, by washing stepwise with decreasing concentrations of aqueous lower alcohols. Ethanol is the preferred lower alcohol for rehydration, but other alcohols may be used. Non-limiting examples include methanol, isopropanol, and other C1-C5 alcohols. Alternatively, the sample is vigorously mixed with the alcohol solution before being removed. In some embodiments, deparaffinization and rehydration are performed simultaneously using a reagent such as, for example, EZ-DEWAX™ (BioGenex, San Ramon, CA).

[0055] In some embodiments, the concentration of alcohol is decreased stepwise. In some embodiments, the concentration range of alcohol in water is decreased stepwise from about 100% to about 70% over about 3 to 5 incremental steps. In some embodiments, the concentration range of alcohol is decreased stepwise over three incremental steps of 100%, 90%, and 70%, respectively. 3. Optional pretreatment for antigen retrieval

[0056] In some embodiments of the present disclosure, samples can be pretreated to, for example, directly or indirectly facilitate the methods of the present invention. In some embodiments, tissue pretreatment increases the availability of target nucleic acids or other targets (e.g., for cell morphology staining). Pretreatment to make targets available (e.g., "antigen retrieval" to retrieve or unmask biological markers of interest). An extensive review of antigen retrieval can be found in Shi et al. 1997, J Histochem Cytochem, 45(3):327. Antigen retrieval includes a variety of methods by which target availability is maximized for interaction with specific detection reagents.

[0057] The most common techniques are protease-induced epitope retrieval (PIER) or heat-induced epitope retrieval (HIER). Protease-induced epitope retrieval (PIER) can use enzymes such as proteinase K, pepsin, trypsin, proteases, and any subtypes thereof in a suitable buffer to restore epitopes for antibody binding. Heat-induced epitope retrieval (HIER) uses heat to reverse some crosslinks and restore epitopes. Citrate buffer, Tris and EDTA base can be used as exemplary heat-inducing reagents in a suitable pH-stabilized format (e.g., 10 mM sodium citrate, pH 6.0; 1 mM EDTA, pH 8.0; 10 mM Tris base, 1 mM EDTA solution, 0.05% Tween 20, pH 9.0). A detergent (e.g., Tween 20) may be added to the HIER buffer to increase epitope retrieval. In certain embodiments, a number of proprietary formulations may be available for PIER or HIER-mediated antigen retrieval.

[0058] For detection of biological markers and identification of cell types, selective staining can be performed on tissue sections (e.g., nuclear and / or cell morphology staining). To facilitate specific recognition of biological markers in fixed tissues (e.g., FFPE tissue samples after deparaffinization and rehydration), retrieval or unmasking of the biological markers of interest is often required via "antigen retrieval" (also called epitope retrieval or antigen unmasking). II. Target immobilization

[0059] It should be understood that embedding of the tissue sample in the gel matrix and immobilization of the nucleic acid target (i.e., covalent attachment of the anchoring agent to the polymer gel) can be performed in any suitable order in various embodiments, so long as they are completed before removing the tissue sample (non-immobilized target nucleic acid) in the gel matrix. It should also be understood that addition and hybridization of the primary probe occurs after the removal step. In some embodiments, the anchoring agent and the primary oligonucleotide probe are not added at the same time and / or in the same step. For example, immobilization of the target nucleic acid can occur before or during embedding of the sample, but the primary oligonucleotide probe is added after the non-target cellular components are removed or removed from the polymer gel matrix. It is understood that immobilization of the target nucleic acid is a multi-step process in which an anchoring agent is first added to the tissue sample, a covalent bond is formed between a first anchoring agent and the target nucleic acid (as disclosed herein for the first anchoring agent of the method), a second anchoring agent comprises an oligonucleotide that hybridizes to the target nucleic acid (as disclosed herein for the anchoring probe or second anchoring agent of the method) and then contacts the polymer matrix, and both the first and second anchoring agents form covalent bonds with the polymer gel. The complete process immobilizes the target nucleic acid in the polymer gel matrix.

[0060] In some embodiments, the target nucleic acid anchoring agent reacts to form a covalent bond with the polymer gel before, during, or after formation of the polymer matrix. A. Anchoring agent

[0061] In some embodiments, at least two anchoring agents are provided for immobilizing a target nucleic acid (e.g., an RNA transcript) to a polymer matrix, as described below. In one embodiment, the first anchoring agent is functionalized to include a first chemical moiety or reactive group that forms a covalent bond with the target nucleic acid, and a second chemical moiety or reactive group that forms a covalent bond with the polymer gel matrix. In another embodiment, the second anchoring agent includes an oligonucleotide that hybridizes with the target nucleic acid, and a chemical moiety or reactive group that forms 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 from 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 some embodiments, the oligonucleotide portion of the second anchoring agent includes poly-T (thymine residues) for hybridizing with the poly-A tail of the mRNA transcript.

[0062] In some embodiments, the anchor probes may comprise sequences complementary to desired (target) nucleic acid species, e.g., sequences that bind (hybridize) thereto via base pairing. In embodiments, the anchor probes comprise chemical moieties or reactive groups that can polymerize (e.g., covalently bond) with the polymer gel matrix.

[0063] In one set of embodiments, the anchoring agent forms a covalent bond with the polymer gel matrix during the polymerization process.For example, in the case of polyacrylamide, the anchoring agent can include an acryldite moiety that can be polymerized and incorporated into the polymer.In certain embodiments, the second anchoring agent or anchor probe includes an oligonucleotide (polyT) that hybridizes with the polyA tail of mRNA and an acryldite moiety that forms a covalent bond with polyacrylamide, and the gel embedding step utilizes polyacrylamide.

[0064] The anchoring agent may also include a moiety that can interact with and bind to a nucleic acid molecule or other molecules that are desired to be immobilized, such as proteins or lipids, other desired targets, etc. The immobilization may be covalent or non-covalent. For example, to immobilize a target nucleic acid, the anchoring agent may include a nucleic acid that includes an acrydite moiety (e.g., at the 5' end, 3' end, internal bases, etc.) and a nucleic acid sequence that is substantially complementary to at least a portion of the target nucleic acid. For example, 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 one, two or more mismatches. In some cases, for example, in the case of transcriptome analysis, the anchoring agent may be configured to immobilize mRNA. For example, in one set of embodiments, the anchoring agent may comprise multiple thymine nucleotides, for example consecutively, to bind to the polyA tail of mRNA. Thus, for example, the anchoring agent may 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., poly dT moieties) 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, locked and non-locked nucleotides may alternate. Such locked thymine nucleotides may be useful, for example, to stabilize the hybridization of the polyA tail of mRNA with the anchoring agent.

[0065] In some embodiments, the method herein further comprises the use of another anchoring agent, referred to herein as the first anchoring agent, which is functionalized with a first and a second chemical moiety for covalent attachment to the target nucleic acid and the polymer gel matrix. In certain embodiments, the anchoring agent is a derivatized alkylating agent that is derivatized with a chemical moiety or reactive group that forms a covalent bond with the polymer gel matrix. Alkylating agents are well known in the art and form covalent bonds with nucleic acids, including RNA, any of which can be derivatized to form the present anchoring agent. In certain embodiments, the anchoring agent is an alkylating agent that is derivatized with a reactive group that forms a covalent bond with polyacrylamide. In certain embodiments, the anchoring agent is an alkylating agent that includes an acrydite moiety. In some 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.

[0066] In some embodiments, the method uses both an anchor probe and an anchoring agent (first and second anchoring agents) to immobilize the target nucleic acid in the polymer gel matrix. In some embodiments, the nucleic acid target is immobilized within the gel via both the anchor probe and the anchoring agent bound to the nucleic acid target.

[0067] In one set of embodiments, the nucleic acid molecule can be immobilized by covalent bonding. For example, in one set of embodiments, an alkylating agent can be used that contains a second chemical moiety that can be covalently attached to the nucleic acid molecule and incorporated into polyacrylamide upon polymerization. In yet another set of embodiments, the terminal ribose in the RNA molecule can be oxidized using sodium periodate (or another oxidizing agent) to generate an aldehyde that can be crosslinked to acrylamide or other polymers or gels. In other embodiments, chemical agents that can modify bases, such as aldehydes, such as paraformaldehyde or glutaraldehyde, alkylating agents or succinimidyl-containing groups; chemical agents that modify terminal phosphates, such as carboimides, such as EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide); chemical agents that modify internal sugars, such as p-maleimido-phenylisocyanate; or chemical agents that modify terminal sugars, such as sodium periodate, can be used. In some cases, these chemical agents can have a second chemical moiety that can be directly crosslinked to the gel or polymer and / or can be further modified with a compound that can be directly crosslinked to the gel or polymer.

[0068] In yet other embodiments, the nucleic acid may be immobilized using an anchor probe having a portion that is substantially complementary 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. B. Gel polymer matrix

[0069] In some embodiments, the methods disclosed herein further comprise immobilizing a nucleic acid target binding anchor probe to at least a portion of the tissue sample within the polymer gel.

[0070] The sample may be embedded in a matrix that immobilizes the nucleic acid target. For example, the matrix may include a gel or a polymer, such as polyacrylamide. Thus, for example, acrylamide and a suitable crosslinker (e.g., N,N'-methylenebisacrylamide) may be added to the sample and polymerized to form a gel. The anchor probe, if present, may include a portion that can polymerize with the gel (e.g., an acrydite portion) during the polymerization process, and the nucleic acid (e.g., an mRNA containing a poly-A tail) may then be able to associate with the anchor portion. In this manner, the mRNA may be immobilized in the polyacrylamide gel. As another example, DNA and / or RNA molecules may be immobilized in the polyacrylamide gel using an anchor probe that has a portion that is substantially complementary to the DNA or RNA. As yet another example, the DNA and / or RNA molecules may be physically entangled in the polyacrylamide gel, for example, by their length, to immobilize them in the polyacrylamide gel.

[0071] The sample may be partially or completely immobilized or embedded in a polymer or gel. In some cases, the sample may be embedded in a relatively large polymer or gel, which in some cases can then be sectioned or sliced, for example, using various microtome sectioning techniques commonly available to those skilled in the art, to create smaller pieces for analysis. For example, tissues or organs can be immobilized in a suitable polymer or gel.

[0072] In some embodiments, a variety of polymers can be used. In some cases, the polymer can be selected to be relatively optically transparent. The polymer can 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 can be determined as a relative change in size, 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 the length dimension of the sample doubles), or the reciprocal of these (i.e., an inverse change in size of 2 means that the sample halves in the length dimension).

[0073] Examples of suitable polymers include polyacrylamide and agarose. In some embodiments, the polymer is not a hydrogel and / or does not include polymers or monomers that swell or expand. In various embodiments that include chemical crosslinks between gel subunits, various polymers can be used, including, but not limited to, acrylic acid, acrylamide, ethylene glycol diacrylate, ethylene glycol dimethacrylate, poly(ethylene glycol dimethacrylate); and / or hydrophobic or hydrogen bonding interactions, such as poly(N-isopropylacrylamide), methylcellulose, (ethylene oxide)-(propylene oxide)-(ethylene oxide terpolymers, sodium alginate, poly(vinyl alcohol), alginates, chitosan, gum arabic, gelatin, and agarose. III. Tissue Removal

[0074] After the nucleic acid targets are immobilized in the gel, other components in the sample that are not the desired target (eg, non-immobilized target nucleic acid) can be removed or degraded.

[0075] By "removing" a tissue sample or a "removed" tissue sample, it is meant that the tissue sample is made substantially transparent, i.e., transparent, to light, and the optical properties of the sample are changed 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) used to illuminate the sample passes through the sample, illuminating 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 of the light, 98% or more of the light, e.g., 100% of the light passes through the specimen. Any process known for tissue removal can be used to remove tissue samples in the methods described herein, which are further described below.

[0076] Details of tissue clearing are further discussed in U.S. Patent Application Publication No. 2019 / 0264270, entitled "Matrix imprinting and clearing," published August 29, 2019, the entire contents of which are incorporated herein by reference. Such clearing may include removing (e.g., physically removing) and / or degrading cellular components from the sample such that they are no longer noticeable in the background. Degradation may include, for example, chemical degradation, enzymatic degradation, etc.

[0077] In some cases, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the undesired components in the sample may be removed or decomposed. Such removal may include physical removal or decomposition of components (e.g., smaller components, non-fluorescent constituents, etc.). Such removal or decomposition of components may reduce background fluorescence or autofluorescence in the sample being analyzed.

[0078] In certain embodiments, multiple removal steps may be performed, for example, to remove or decompose various undesirable components.

[0079] For example, enzymes, denaturants, chelating agents, chemical agents, etc. can break down proteins into smaller components and / or amino acids. These smaller components can be easily physically removed and / or be small or inert enough that they do not significantly affect the background. Similarly, lipids can be removed or decomposed from the sample using detergents, etc. In some cases, one or more of these are used, for example, simultaneously or sequentially. Non-limiting 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 HCl, acetone, acetic acid, urea, or lithium perchlorate. Non-limiting examples of chemical agents that can denature proteins include solvents, such as phenol, chloroform, guanidinium isocyanate, urea, formamide, etc. Non-limiting examples of detergents include Triton X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether), SDS (sodium dodecyl sulfate), Igepal CA-630, or poloxamer. Non-limiting examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), citrate, or polyaspartic acid. In some embodiments, compounds such as these can be applied to a sample to remove or degrade proteins, lipids, and / or other components. For example, a buffer (e.g., containing Tris or tris(hydroxymethyl)aminomethane) can be applied to the sample and then removed.

[0080] Non-limiting examples of techniques for removing or degrading RNA include RNA enzymes such as Rnase A, Rnase T, or Rnase H, or chemical agents, for example, by alkaline hydrolysis (e.g., by raising the pH to above 10). Non-limiting examples of systems for removing or degrading sugars or extracellular matrix include enzymes such as chitinase, heparinase, or other glycosylases. Non-limiting examples of systems for removing or degrading lipids include enzymes, such as lipidases, chemical agents, 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 can be reduced, which can facilitate analysis of nucleic acid probes or other desired targets, for example, using fluorescence microscopy or other techniques discussed herein. IV. Formation of Nucleic Acid Target / Probe Complexes A. Nucleic acid target

[0081] The nucleic acid target can be, for example, DNA, RNA, or other nucleic acid present in cells within a tissue sample.

[0082] In some embodiments, the nucleic acid target is RNA.RNA can be coding RNA and / or non-coding RNA.Non-limiting examples of RNA that can be studied in cells include mRNA, siRNA, rRNA, miRNA, tRNA, lncRNA, snoRNA, snRNA, exRNA, piRNA, etc.

[0083] The nucleic acid may be endogenous to the cell or may be added to the cell. For example, the nucleic acid may be viral or artificially created. In some cases, the nucleic acid to be determined may be expressed by the cell.

[0084] In some cases, a significant portion of the nucleic acids in a cell can be studied. For example, in some cases, sufficient RNA present in the cell can be determined to generate a partial or complete transcriptome of the cell. In some cases, at least four unique mRNA gene transcripts are determined in the 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 25 6. 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, at least 100,000 types of mRNA can be determined in a cell.

[0085] In some cases, the transcriptome of a cell can be determined. It should be understood that the transcriptome generally encompasses all RNA transcript molecules produced in the cell, both coding and non-coding, not just coding messenger RNA. Thus, for example, the transcriptome can 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 can be determined.

[0086] The determination of one or more nucleic acids in a sample can be qualitative and / or quantitative. Furthermore, the determination can be spatial, for example, the location of the nucleic acid in the sample can be determined in two or three dimensions. In some embodiments, the location, number, and / or concentration of the nucleic acid in a cell (or other sample) can be determined. B. Nucleic Acid Probes

[0087] In one set of embodiments, as an illustrative, non-limiting example, the removed non-target cellular components and fixed target nucleic acid sample ("matrix-anchored target nucleic acid sample") can be studied by exposing it to one or more types of primary oligonucleotide nucleic acid probes and imaged simultaneously or sequentially using a secondary nucleic acid probe (e.g., fluorescently labeled).

[0088] For example, in one set of embodiments, the nucleic acid probes can include smFISH probes or MERFISH probes, such as those discussed in U.S. Pat. No. 11,098,303 or U.S. Pat. No. 10,240,146, respectively, which are incorporated by reference in their entireties.

[0089] The nucleic acid probe may comprise a nucleic acid (or an entity that can hybridize, e.g., specifically, to a nucleic acid), such as DNA, RNA, LNA (locked nucleic acid), PNA (peptide nucleic acid), or a combination thereof. In some cases, additional components may also be present within the nucleic acid probe, e.g., as discussed below. The nucleic acid probe may be introduced into the sample using any suitable method.

[0090] The nucleic acid probe is added to the sample containing the gel-immobilized target nucleic acid after non-target cellular components have been removed from the gel. Certain aspects of the invention generally relate to a nucleic acid probe being introduced into the sample. The probe may comprise any of a variety of entities, such as DNA, RNA, LNA, PNA, etc., that can hybridize to a nucleic acid, typically by Watson-Crick base pairing, depending on the application. The nucleic acid probe typically comprises a target sequence that can bind, possibly specifically, to at least a portion of the target nucleic acid. Once introduced into the sample, the nucleic acid probe may be capable of binding to a specific target nucleic acid (e.g., mRNA or other nucleic acid discussed herein). In some cases, the nucleic acid probe may be determined using a signaling entity (e.g., as described below) and / or by using a secondary nucleic acid probe that can bind to the nucleic acid probe (i.e., the primary nucleic acid probe). Such determination of the nucleic acid probe is discussed in detail below.

[0091] In some cases, two or more different (primary) nucleic acid probes can be applied to the sample, e.g., simultaneously. For example, there can 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 applied, e.g., simultaneously or sequentially, to the sample.

[0092] In certain embodiments, the primary oligonucleotide probe comprises a target sequence designed to hybridize with the anchored target nucleic acid. The target sequence can be located anywhere within the nucleic acid probe (or the primary nucleic acid probe or the coding nucleic acid probe). The target sequence can comprise a region that is substantially complementary to a portion of the target nucleic acid. In some cases, the portion can 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 100% complementary. In some cases, the length of the target sequence can 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 some cases, the length of the target sequence can be 500 nucleotides or less, 450 nucleotides or less, 400 nucleotides or less, 350 nucleotides or less, 300 nucleotides or less, 250 nucleotides or less, 200 nucleotides or less, 175 nucleotides or less, 150 nucleotides or less, 125 nucleotides or less, 100 nucleotides or less, 75 nucleotides or less, 60 nucleotides or less, 65 nucleotides or less, 60 nucleotides or less, 55 nucleotides or less, 50 nucleotides or less, 45 nucleotides or less, 40 nucleotides or less, 35 nucleotides or less, 30 nucleotides or less, 20 nucleotides or less, or 10 nucleotides or less. Any combination of these is also possible, for example, the target sequence may have a length of 10-30 nucleotides, 20-40 nucleotides, 5-50 nucleotides, 10-200 nucleotides, or 25-35 nucleotides, 10-300 nucleotides, etc. Typically, complementarity is determined based on Watson-Crick nucleotide base pairing.

[0093] The target sequence of the (primary) nucleic acid probe may be determined with reference to a target nucleic acid suspected to be present in the sample. For example, the target nucleic acid for a protein may be determined using the sequence of the protein by determining the nucleic acid expressed to form the protein. In some cases, only a portion of the nucleic acid encoding the protein, e.g., having a length as described above, is used. In addition, in some cases, two or more target sequences may be used that can be used to identify a specific target. For example, multiple probes that can bind or hybridize to different regions of the same target may be used sequentially and / or simultaneously. Hybridization typically refers to the annealing process in which complementary single-stranded nucleic acids associate to form double-stranded nucleic acids via Watson-Crick nucleotide base pairing (e.g., hydrogen bonds, guanine-cytosine and adenine-thymine).

[0094] In some embodiments, a nucleic acid probe, such as a primary nucleic acid probe, may also include one or more "lead" sequences designed to hybridize with a secondary nucleic acid probe that includes a label (e.g., a fluorescent label). However, it should be understood that a lead sequence is not necessary in all cases. In some embodiments, a nucleic acid probe may include 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 lead sequences. The lead sequences may be located anywhere within the nucleic acid probe. When multiple lead sequences are present, the lead sequences may be located adjacent to each other and / or may be interspersed with other sequences. In certain embodiments, a primary oligonucleotide probe includes one lead sequence. In certain other embodiments, a primary oligonucleotide probe includes two lead sequences that may be the same or different from each other (meaning, for example, that the secondary nucleic acid probe does not hybridize to different lead sequences).

[0095] Lead sequence, if present, can be any length.When two or more lead sequences are used, the lead sequences can have the same or different length independently.For example, the length of lead sequence can 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 some cases, the length of the lead sequence can be 500 nucleotides or less, 450 nucleotides or less, 400 nucleotides or less, 350 nucleotides or less, 300 nucleotides or less, 250 nucleotides or less, 200 nucleotides or less, 175 nucleotides or less, 150 nucleotides or less, 125 nucleotides or less, 100 nucleotides or less, 75 nucleotides or less, 60 nucleotides or less, 65 nucleotides or less, 60 nucleotides or less, 55 nucleotides or less, 50 nucleotides or less, 45 nucleotides or less, 40 nucleotides or less, 35 nucleotides or less, 30 nucleotides or less, 20 nucleotides or less, or 10 nucleotides or less. Any combination of these is also possible, for example, the lead sequence can have a length of 10-30 nucleotides, 20-40 nucleotides, 5-50 nucleotides, 10-200 nucleotides, or 25-35 nucleotides, 10-300 nucleotides, etc.

[0096] In some embodiments, the lead sequence may be arbitrary or random. In certain cases, the lead sequence is selected to reduce or minimize homology with other components of the sample, for example, so that the lead sequence itself does not bind or hybridize to other nucleic acids suspected to be in 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 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 of homology. In some cases, the base pairs are consecutive.

[0097] In certain embodiments, the primary oligonucleotide probes are provided as a pool of probes, with each pool of nucleic acid probes hybridizing to a different target nucleic sequence (e.g., a different RNA transcript). In some embodiments, each pool of probes encodes an N-bit binary code assigned to each distinct RNA transcript via the lead sequence. 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, where the Hamming weight value is the number of "1" values ​​in the N-bit code, with all other positions being "0". In some embodiments, the N-bit binary code has a Hamming weight of at least 2 or at least 4, meaning that the code contains two or four "1" bit values, respectively, with the other bit positions being "0". In some embodiments, the N-bit binary code has an N value between 3 and 100, with any value possible. In certain embodiments, the binary code is a 4-bit binary code, a 6-bit binary code, an 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", and the binding of the secondary probe to the read sequence determines whether the probe is not bound, which is a "0", or the secondary probe is bound to the read sequence of the primary probe, which is a "1". Successive hybridizations and imaging of the secondary read probes are performed until each position of the N-bit binary code is read to obtain a barcode or codeword of the target nucleic acid (e.g., an mRNA sequence).

[0098] In one set of embodiments, a population of nucleic acid probes may contain a certain number of lead sequences, which in some cases may be less than the number of targets of the nucleic acid probe. Those skilled in the art will recognize that when there is one signaling entity and n lead sequences, typically 2n-1 different nucleic acid targets may be uniquely identified. However, not all possible combinations need to be used. For example, a population of nucleic acid probes may target 12 different nucleic acid sequences but contain 8 or fewer lead sequences. As another example, a population of nucleic acids may target 140 different nucleic acid species but contain 16 or fewer lead sequences. Different nucleic acid sequence targets can be separately identified by using different combinations of lead sequences within each probe. For example, each probe may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc., or more lead sequences. In some cases, a population of nucleic acid probes may each contain the same number of lead sequences, while in other cases, there may be different numbers of lead sequences on the various probes.

[0099] As a non-limiting example, a first nucleic acid probe may include a first target sequence, a first lead sequence and a second lead sequence, and a second, different nucleic acid probe may include a second target sequence, the same first lead sequence, but instead of the second lead sequence, a third lead sequence, such probes may be identified by determining that various lead sequences are present or associated with a given probe or location, as discussed herein.

[0100] Additionally, nucleic acid probes (and their corresponding complementary sites on the code probe) may, in certain embodiments, be made using only two or only three of the four bases, for example, by excluding all Gs or excluding all Cs in the probe. In certain embodiments, sequences lacking either Gs or Cs may be less able to form secondary structures, contributing to more uniform and faster hybridization.

[0101] In some embodiments, the nucleic acid probe may include a signaling entity. However, it should be understood that a signaling entity is not required in all cases. For example, the nucleic acid probe may be determined in some embodiments using a secondary nucleic acid probe, as described in more detail below. Examples of signaling entities that may be used are also discussed in more detail below.

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

[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 a polymerase enzyme, such as a nucleic acid polymerase, to extend the primer and replicate the complementary strand. The primer is complementary to and hybridizes to (e.g., is designed to be) a target nucleic acid. In some embodiments, the primer is a synthetic primer. In some embodiments, the primer is a non-natural primer. The primer typically has a length of 10 to 50 nucleotides. For example, the primer can 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, the primer has a length of 18 to 24 nucleotides.

[0104] Furthermore, the components of the nucleic acid probe can be arranged in any suitable order. For example, in one embodiment, the components can be arranged in the nucleic acid probe as primer-lead sequence-target sequence-lead sequence-reverse primer. The "lead sequences" in this structure can each contain any number of lead sequences (including 0), so long as there is at least one lead sequence in the probe. Non-limiting exemplary structures include the following: · Primer-target sequence-read sequence-reverse primer Primer-Read sequence-Target sequence-Reverse primer · Target sequence-primer-target sequence-read sequence-reverse primer · Target sequence-primer-read sequence-target sequence-reverse primer Primer-target sequence-read sequence-target sequence-reverse primer · Target sequence-Primer-Read sequence-Reverse primer Target sequence - Lead sequence - Primer Lead sequence - Target sequence - Primer · Lead sequence-primer-target sequence-reverse primer, etc. Additionally, the reverse primer is optional in some embodiments, including all of the above examples. V. Detection / imaging of nucleic acid target / probe complexes

[0105] After the primary nucleic acid probe is introduced into the sample, according to certain aspects of the invention, the nucleic acid probe can be determined directly by determining the signaling entity (if present) and / or by using one or more secondary nucleic acid probes (also referred to herein as readout probes). As mentioned, in some cases, the determination can be spatial, e.g., two-dimensional or three-dimensional. Furthermore, in some cases, the determination can be quantitative, e.g., the amount or concentration of the primary nucleic acid probe (and the target nucleic acid) can be determined. Furthermore, the secondary probe can include any of a variety of entities that can hybridize to nucleic acids, e.g., DNA, RNA, LNA and / or PNA, depending on the application. Signaling entities are described in more detail below.

[0106] The secondary nucleic acid probe may contain a recognition sequence that can bind or hybridize to the lead sequence of the primary nucleic acid probe. In some cases, the binding may be specific, or the binding may be such that the recognition sequence preferentially binds or hybridizes to only one of the lead sequences present. The secondary nucleic acid probe may also contain one or more signaling entities. When two or more secondary nucleic acid probes are used, the signaling entities may be the same or different. In an embodiment, the secondary nucleic acid probe contains a fluorescent label and may be referred to herein as a fluorescent secondary nucleic acid probe.

[0107] The recognition sequence may be of any length, and multiple recognition sequences may be the same or different lengths. When more than one recognition sequence is used, the recognition sequences may independently have the same or different lengths. For example, 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 long. In some cases, the recognition sequence may be 75 or less, 60 or less, 65 or less, 60 or less, 55 or less, 50 or less, 45 or less, 40 or less, 35 or less, 30 or less, 20 or less, or 10 or less nucleotides long. Any combination of these is also possible, for example, the recognition sequence may have a length of 10-30, 20-40, or 25-35 nucleotides, etc. In one embodiment, the recognition sequence is the same length as the lead sequence. Additionally, 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 the lead sequence of the primary nucleic acid probe.

[0108] As mentioned, in some cases, the secondary nucleic acid probe may include one or more signaling entities, examples of which are described in more detail below.

[0109] As discussed, certain aspects of the invention use nucleic acid probes that include various "lead sequences." For example, a population or pool of primary nucleic acid probes can include specific "lead sequences" that can bind to specific secondary nucleic acid probes, and the location of the primary nucleic acid probes is determined within a sample using, for example, a secondary nucleic acid probe that includes a signaling entity. As mentioned, in some cases, a population of lead sequences can be combined in various combinations to create different nucleic acid probes, such that, for example, a relatively small number of lead sequences can be used to create a relatively large number of different nucleic acid probes.

[0110] Thus, in some cases, each population of primary nucleic acid probes (or other nucleic acid probes) may contain a certain number of lead sequences, some of which may be shared between different primary nucleic acid probes, such that the entire population of primary nucleic acid probes may contain a certain number of lead sequences. A population of nucleic acid probes may have any suitable number of lead 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. lead sequences. In some embodiments, more than 20 are possible. Additionally, in some cases, a population of nucleic acid probes may have, in total, 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 lead sequence occurrences, although some or all of the probes may each have two or more lead sequences, as discussed herein. Additionally, in some embodiments, the population of nucleic acid probes may have 100 or less, 80 or less, 64 or less, 60 or less, 50 or less, 40 or less, 32 or less, 24 or less, 20 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less lead sequences. Any combination of these is also possible, for example, the population of nucleic acid probes may include a total of 10-15 lead sequences.

[0111] 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 lead sequences, in a population of six different types of nucleic acid probes, each containing one or more lead sequences, the total number of lead sequences in the population may be four or less. While four lead sequences are used in this example for ease of illustration, it should be understood that in other embodiments, a greater number of nucleic acid probes may be achieved using, for example, 5, 8, 10, 16, 32, etc., or more lead sequences, or any other suitable number of lead sequences described herein, depending on the application. If each of the primary nucleic acid probes contains two different lead sequences, up to six probes can be individually identified by using four such lead sequences (A, B, C, and D). Note that in this example, ordering of the lead sequences on the nucleic acid probe is not required, i.e., "AB" and "BA" may be treated as synonymous (however, in other embodiments, the order of the lead sequences may be required and "AB" and "BA" may not necessarily be synonymous). Similarly, if five lead sequences (A, B, C, D, and E) are used in a population of primary nucleic acid probes, up to 10 probes can be identified separately. For example, those skilled in the art will understand that for k lead sequences in a population with n lead sequences on each probe, up to x different probes can be made, assuming that ordering of the lead sequences is not essential. Since not all probes need to have the same number of lead sequences, and not all combinations of lead sequences need to be used in all embodiments, more or less different probes may be used in certain embodiments. Furthermore, it should also be understood that in some embodiments, the number of lead sequences on each probe need not be the same. For example, some probes may contain two lead sequences, while other probes may contain three lead sequences.

[0112] In some embodiments, the lead sequence and / or binding pattern of the nucleic acid probes in the sample can be used to define an error detection and / or error correction code, for example, to reduce or prevent misidentification or errors of the nucleic acid. Thus, when using a primary nucleic acid probe pool that includes lead sequences, for example, if binding is indicated (e.g., determined using a signaling entity), the location can be identified with a "1" and, conversely, if binding is not indicated, the location can be identified with a "0" (and vice versa in some cases). Here, each probe pool encodes an N-bit binary code with a Hamming weight of at least 2 assigned to each distinct target nucleic acid (e.g., RNA transcript) via the lead sequence. Then, for example, multiple rounds of binding determination using different nucleic acid probes can be used to create a "code word" for that spatial location, for example, based on the binding of the readout probe to the lead sequence of the primary probe. In some embodiments, the N-bit binary code can be subject to error detection and / or correction. For example, the code word can be organized such that if no match is found for a given set of lead sequences or binding patterns of nucleic acid probes, the match can be identified as an error and, optionally, error correction can be applied to determine the correct target of the nucleic acid probe. In some cases, a codeword may have fewer "letters" or positions than the total number of nucleic acids encoded by the codewords, for example, if each codeword encodes a different nucleic acid.

[0113] Such error detection and / or error correction codes can take a variety of forms. A variety of such codes have been previously developed in other contexts, such as the telecommunications industry, such as Golay or Hamming codes. In one set of embodiments, the read sequences or binding patterns of the nucleic acid probes are assigned such that all possible combinations are not assigned.

[0114] For example, if four lead sequences are possible and the primary nucleic acid probe contains two lead sequences, then up to six primary nucleic acid probes may be identified, but the number of primary nucleic acid probes used may be less than six. Similarly, for k lead sequences in a population with n lead sequences on each primary nucleic acid probe, different probes may be made, but the number of primary nucleic acid probes used may be more or less than k. Furthermore, these may be assigned randomly or in a specific manner to increase the ability to detect and / or correct errors.

[0115] As another example, when multiple rounds of nucleic acid probes are used, the number of rounds can be selected arbitrarily. If each target can have two possible outcomes, such as being detected or not detected, in each round, up to 2n different targets can be possible for the probes in n rounds, but the number of nucleic acid targets actually used can be any number less than 2n. For example, if each round can have three or more possible outcomes, such as each target being detected in a different color channel, more than 2n (e.g., 3n, 4n...) different targets can be possible for the n rounds of probes. In some cases, the number of nucleic acid targets actually used can be any number less than this number. Furthermore, they can be randomly assigned or assigned in a specific manner to increase the ability to detect and / or correct errors.

[0116] For example, in one set of embodiments, code words or nucleic acid probes can be assigned in the code space to be separated by a Hamming distance, thereby measuring the number of erroneous "reads" in a given pattern that mislead the nucleic acid probes into being different valid nucleic acid probes. The Hamming weight refers to the distance between the N-bit binary code assigned to the target and each pool of primary oligonucleotide probes thus coded via their read sequences. In some embodiments, the pool of primary probes can have an assigned N-bit binary code with a Hamming weight of at least 4 and a Hamming weight of 4 between the pools. In that embodiment, both error detection and correction are possible. In some cases, the Hamming distance may be at least 2, at least 3, at least 4, at least 5, at least 6, etc. Additionally, in one set of embodiments, the assignments can be formed as Hamming codes, such as Hamming (7,4) codes, Hamming (15,11) codes, Hamming (31,26) codes, Hamming (63,57) codes, Hamming (127,120) codes, etc. In other sets of embodiments, the assignments may form a SECDED code, such as a SECDED(8,4) code, a SECDED(16,4) code, a SCEDED(16,11) code, a SCEDED(22,16) code, a SCEDED(39,32) code, a SCEDED(72,64) code, etc. In yet other sets of embodiments, the assignments may form an extended binary Golay code, a full binary Golay code, or a ternary Golay code. In another set of embodiments, the assignments may represent a subset of the possible values ​​obtained from any of the above-mentioned codes.

[0117] For example, by using only binary words containing a fixed number of "1" bits, e.g., 4, to code the target, a code with the same error correction properties of the SECDED code can be formed. In another set of embodiments, the assignments can represent a subset of the possible values ​​obtained from the above codes, with the purpose of addressing asymmetric read errors. For example, in some cases, a code that can fix the number of "1" bits for all binary words used can eliminate biased measurements of words with different numbers of "1" when the rate at which "0" bits are measured as "1" or "1" bits are measured as "0" is different.

[0118] Thus, in some embodiments, once a codeword is determined (e.g., as described herein), the codeword can be compared to 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 reading the codeword can be identified. In some cases, error correction can also be applied to determine the correct codeword, and thus the correct identity of the nucleic acid target. In some cases, the codeword can be selected such that, assuming there is only one error, only one possible correct codeword is available, and thus only one correct identity of the nucleic acid target is possible. In some cases, this can also be generalized to larger codeword intervals or Hamming distances. For example, if there are two, three, or four errors (or possibly more), then the codeword can be selected such that only one possible correct codeword is available, and thus only one correct identity of the nucleic acid target is possible.

[0119] The error correction code may be a binary error correction code, or may be based on other numbering systems, such as ternary or quaternary error correction codes. For example, in one set of embodiments, two or more types of signaling entities may be used and assigned different numbers in the error correction code. Thus, as a non-limiting example, a first signaling entity (possibly two or more signaling entities) may be assigned as "1" and a second signaling entity (possibly two or more signaling entities) may be assigned as "2" (with "0" indicating the absence of a signaling entity), and a code word defining a ternary error correction code is dispensed. Similarly, a third signaling entity may be further assigned as "3" to create a quaternary error correction code, and so on.

[0120] The contents of each of the following references are incorporated herein by reference for further discussion of multiplex error-robust fluorescent in situ hybridization (MERFISH) and examples thereof (e.g., MERFISH probes, signal amplification, determination of nucleic acid probes, codeword creation, and error detection and correction, etc.) for further discussion of multiplex error-robust fluorescent in situ hybridization (MERFISH) and examples thereof (e.g., MERFISH probes, signal amplification, determination of nucleic acid probes, codeword creation, and error detection and correction, etc.) described herein.

[0121] As discussed above, in certain aspects, the signaling entities are determined, e.g., to determine a nucleic acid probe and / or to create a codeword. In some cases, the signaling entities within the sample may be determined, e.g., spatially, using a variety of techniques. In some embodiments, the signaling entities may be fluorescent, and the location of the signaling entities within the cell may be spatially identified using techniques for determining fluorescence within the sample, such as fluorescence microscopy or confocal microscopy. In some cases, the location of the entities within the sample may be determined in two or even three dimensions. Additionally, in some embodiments, more than one signaling entity may be determined at once (e.g., signaling entities having different colors or emissions) and / or sequentially.

[0122] Furthermore, in some embodiments, a confidence level for the identified nucleic acid target can be determined. For example, the confidence level can be determined using the ratio of the number of perfect matches to the number of matches with one or more 1-bit errors. In some cases, only matches with a confidence ratio greater than a certain value can be used. For example, in certain embodiments, a match can 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. Further, in some embodiments, a match can be accepted only if the confidence ratio for the identified nucleic acid target exceeds the 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 value.

[0123] In some embodiments, the spatial location of the entity (and thus the nucleic acid probe with which the entity may be associated) may be determined with a relatively high resolution. For example, the spatial resolution at which the location is determined may be 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.

[0124] There are various techniques by which the spatial location of an entity or target can be determined or imaged optically, for example using fluorescence microscopy, using radioactivity, by conjugation with a suitable chromophore, or otherwise. For example, various conventional microscopy techniques that may be used in various embodiments of the present invention include, but are not limited to, epifluorescence microscopy, total internal reflection microscopy, highly inclined thin-illumination (HILO) microscopy, light sheet microscopy, scanning confocal microscopy, scanning line confocal microscopy, spinning disk confocal microscopy, or other equivalent conventional microscopy techniques.

[0125] In some embodiments, in situ hybridization (ISH) techniques for labeling nucleic acids such as DNA or RNA can be used, e.g., nucleic acid probes can be hybridized to nucleic acids in a sample. These can be performed, e.g., at a cellular or single molecule scale resolution. In some cases, the ISH probes can be composed of RNA, DNA, PNA, LNA, other synthetic nucleotides, etc., and / or any combination of these. The presence of hybridized probes can be measured, e.g., by radioactivity using radioactively labeled nucleic acid probes, immunohistochemistry using, e.g., biotin-labeled nucleic acid probes, enzyme chromophore or fluorophore generation using probes that can be bound to enzymes such as horseradish peroxidase and approaches such as tyramide signal amplification, fluorescent imaging using nucleic acid probes directly labeled with fluorophores, or hybridization of these primary probes with secondary nucleic acid probes, which are detected via any of the methods described above.

[0126] In some cases, spatial location may be determined with super-resolution, or with a resolution better than the wavelength or diffraction limit of light (although in other embodiments super-resolution is not required). Non-limiting examples include STORM (Stochastic Optical Reconstruction Microscopy), STED (Stimulated Emission Depletion Microscopy), NSOM (Scanning Near-Field 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), Photoactivated Localization Microscopy (PALM), Fluorescence Photoactivated Localization Microscopy (FPALM), LIMON (3D Light Microscopy Nanosizing Microscopy), Super-Resolution Optical Fluctuation Imaging (SOFI), and the like. See, for example, U.S. Patent No. 7,838,302, issued November 23, 2010 to Zhuang, et al., entitled "Sub-Diffraction Limit Image Resolution and Other Imaging Techniques"; U.S. Patent No. 8,564,792, issued October 22, 2013 to Zhuang, et al., entitled "Sub-diffraction Limit Image Resolution in Three Dimensions"; or International Publication No. WO 2013 / 090360, issued June 20, 2013 to Zhuang, et al., entitled "High Resolution Dual-Objective Microscopy," each of which is incorporated herein by reference in its entirety.

[0127] In one embodiment, the sample may be illuminated by a single Gaussian mode laser line. In some embodiments, the profiled illumination can be flattened by passing these laser lines through a multimode fiber that is vibrated via piezoelectric or other mechanical means. In some embodiments, the illumination profile can be flattened by passing the single mode Gaussian beam through various refractive beam shapers, such as a piShaper or a series of stacked Powell lenses. In yet another set of embodiments, the Gaussian beam can be passed through a variety of different diffusing elements, such as ground glass or engineered diffusers, which in some cases can be rotated at high speed to remove residual laser speckle. In yet another embodiment, the laser illumination can be passed through a series of lenslet arrays to generate overlapping images of the illumination that approximate a flat illumination field.

[0128] In some embodiments, the centroid of the spatial location of the entity can be determined. For example, the centroid of a signaling entity can be determined within an image or series of images using image analysis algorithms known to those skilled in the art. In some cases, the algorithm may be selected to determine non-overlapping and / or partially overlapping single emitters within a sample. Non-limiting examples of suitable techniques include maximum likelihood algorithms, least squares algorithms, Bayesian algorithms, compressive sensing algorithms, and the like. In some cases, a combination of these techniques can also be used.

[0129] Furthermore, the signaling entities 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 capable of recognizing a first lead sequence, and then the first secondary nucleic acid probe may be inactivated before a second secondary nucleic acid probe is applied to the sample. When multiple signaling entities are used, the signaling entities may be inactivated using the same or different techniques, and some or all of the multiple signaling entities may be inactivated, for example, sequentially or simultaneously.

[0130] Inactivation can occur by removal of the signaling entity (e.g., from the sample, or from the nucleic acid probe, etc.) and / or by some other way, such as photobleaching, bleaching, or chemically altering the structure of the signaling entity, such as by reduction. For example, in one set of embodiments, a fluorescent signaling entity can be inactivated by chemical or optical techniques, such as oxidation, photobleaching, chemical bleaching, stringent washing or enzymatic digestion or reaction by exposure to enzymes, dissociation of the signaling entity from other components (e.g., probes), chemical reaction of the signaling entity (e.g., with reactants that can change the structure of the signaling entity), etc. For example, bleaching can occur by exposure to oxygen, reducing agents, or the signaling entity can be chemically cleaved from the nucleic acid probe and washed away by a fluid flow.

[0131] In some embodiments, the various nucleic acid probes (including the primary and / or secondary nucleic acid probes) may contain one or more signaling entities. When more than one nucleic acid probe is used, the signaling entities may be the same or different. In certain embodiments, the signaling entity is any entity that can emit light. For example, in one embodiment, the signaling entity is fluorescent. In other embodiments, the signaling entity may be phosphorescent, emissive, absorptive, etc. In some cases, the signaling entity is any entity that can be determined within a sample with relatively high resolution, e.g., better than the wavelength or diffraction limit of visible light. The signaling entity may be, for example, a dye, a small molecule, a peptide, or a protein, etc. The signaling entity may be a single molecule in some cases. When multiple secondary nucleic acid probes are used, the nucleic acid probes may contain the same or different signaling entities.

[0132] Non-limiting examples of signaling entities include fluorescent entities (fluorophores) or phosphorescent entities, such as 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, PSCFP2, Dendra, Dendra2, EosFP, tdEos, mEos2, mEos3, PamCherry, PAtagRFP, mMaple, mMaple2, and mMaple3. Other suitable signaling entities are known to those skilled in the art. See, for example, U.S. Pat. No. 7,838,302 or WO2015160690A1, each of which is incorporated herein by reference in its entirety. In some cases, spectrally distinct fluorescent dyes may be used.

[0133] In one set of embodiments, the signaling entity may be attached to the oligonucleotide sequence via a bond that can be cleaved to release the signaling entity. In one set of embodiments, the fluorophore may be conjugated to the oligonucleotide via a cleavable bond, such as a photocleavable bond. Non-limiting examples of photocleavable bonds include 1-(2-nitrophenyl)ethyl, 2-nitrobenzyl, biotin phosphoramidite, acryl phosphoramidite, diethylaminocoumarin, 1-(4,5-dimethoxy-2-nitrophenyl)ethyl, cyclododecyl(dimethoxy-2-nitrophenyl)ethyl, 4-aminomethyl-3-nitrobenzyl, (4-nitro-3-(1-chlorocarbonyloxyethyl)phenyl)methyl-S-acetylthio acid ester, (4-nitro-3-(1-chlorocarbonyloxyethyl)phenyl)methyl-3-(2-pyridyldithiopropionic acid) ester, 3-(4,4'-dimethoxytrityl)-1-(2-nitrophenyl)-propane-1,3-diol-[2-cyano]-1,2-diphenyl ... Noethyl-(N,N-diisopropyl)]-phosphoramidite, 1-[2-nitro-5-(6-trifluoroacetylcaproamidomethyl)phenyl]-ethyl-[2-cyano-ethyl-(N,N-diisopropyl)]-phosphoramidite, 1-[2-nitro-5-(6-(4,4'-dimethoxytrityloxy)butylamidomethyl)phenyl]-ethyl-[2-cyanoethyl-(N,N-diisopropyl)]-phosphoramidite, 1-[2-nitro-5-(6-(N-(4,4'-dimethoxytrityl))-biotinamidocaproamido-methyl)phenyl]-ethyl-[2-cyanoethyl-(N,N-diisopropyl)]-phosphoramidite, or similar linkers. In another set of embodiments, the fluorophore may be conjugated to the oligonucleotide via a disulfide bond.Disulfide bonds can be cleaved by a variety of reducing agents, including, but not limited to, dithiothreitol, dithioerythritol, beta-mercaptoethanol, sodium borohydride, thioredoxin, glutaredoxin, trypsinogen, hydrazine, diisobutylaluminum hydride, oxalic acid, formic acid, ascorbic acid, phosphorous acid, stannous chloride, glutathione, thioglycolate, 2,3-dimercaptopropanol, 2-mercaptoethylamine, 2-aminoethanol, tris(2-carboxyethyl)phosphine, bis(2-mercaptoethyl)sulfone, N,N'-dimethyl-N,N'-bis(mercaptoacetyl)hydrazine, 3-mercaptopropionate, dimethylformamide, thiopropyl-agarose, tri-n-butylphosphine, cysteine, ferrous sulfate, sodium sulfate, phosphites, hypophosphites, phosphorothioates, and the like, and / or any combination thereof. In another embodiment, the fluorophore may be conjugated to the oligonucleotide via one or more phosphorothioate modified nucleotides, which replace the bridging and / or non-bridging oxygens with sulfur modifications. The fluorophore may be cleaved from the oligonucleotide by the addition of compounds such as, but not limited to, iodoethanol, iodine mixed in ethanol, silver nitrate, or mercuric chloride, in certain embodiments. In yet another set of embodiments, the signaling entity may be chemically inactivated by reduction or oxidation. For example, in one embodiment, sodium borohydride may be used to reduce chromophores such as Cy5 or Cy7 to a stable non-fluorescent state. In yet another set of embodiments, the fluorophore may be conjugated to the oligonucleotide via an azo bond, which may be cleaved with 2-[(2-N-arylamino)phenylazo]pyridine. In yet another set of embodiments, the fluorophore may be conjugated to the oligonucleotide via a suitable nucleic acid segment that may be cleaved upon appropriate exposure to a DNAse, such as an exodeoxyribonuclease or an endo-deoxyribonuclease. Examples include, but are not limited to, DNase I or DNase II.In one set of embodiments, cleavage can occur via a restriction endonuclease. Non-limiting examples of potentially suitable restriction endonucleases include BamHI, BsrI, NotI, XmaI, PspAI, DpnI, MboI, MnlI, Eco57I, Ksp632I, DraIII, AhaII, SmaI, MluI, HpaI, ApaI, BclI, BstEII, TaqI, EcoRI, SacI, HindII, HaeII, DraII, Tsp509I, Sau3AI, PacI, and the like. Over 3000 restriction enzymes have been studied in detail, and over 600 of these are commercially available. In yet another set of embodiments, the fluorophore can be conjugated to biotin, and the oligonucleotide can be conjugated to avidin or streptavidin. The interaction between biotin and avidin or streptavidin conjugates the fluorophore to the oligonucleotide, but upon sufficient exposure to excess, free biotin can "outcompete" the binding, thereby causing cleavage. Additionally, in another set of embodiments, the probes can be removed using a corresponding "toehold probe" that contains the same sequence as the probe, as well as an extra number of bases (e.g., 1-20 extra bases, e.g., 5 extra bases) that are homologous to the coding probe. These probes can remove the labeled readout probe by strand displacement interactions.

[0134] As used herein, the term "light" generally refers to electromagnetic radiation having any suitable wavelength (or equivalently, frequency). For example, in some embodiments, light can include wavelengths in the optical or visual range (e.g., having wavelengths from about 400 nm to about 700 nm, i.e., "visible light"), infrared wavelengths (e.g., having wavelengths from about 300 micrometers to 700 nm), ultraviolet wavelengths (e.g., having wavelengths from about 400 nm to about 10 nm), and the like. In certain cases, as discussed in more detail below, two or more entities can be used, i.e., entities that are chemically, e.g., structurally, different or distinct. In other cases, however, the entities may be chemically identical, or at least substantially chemically identical.

[0135] Another aspect of the invention relates to computer-implemented methods. For example, a computer and / or automated system can be provided that can automatically and / or repetitively perform any of the methods described herein. As used herein, an "automated" device refers to a device that can operate without human direction, i.e., an automated device can perform a function for a period of time after someone has finished performing some action to facilitate the function, e.g., by inputting instructions to a computer to start a process. Typically, an automated device can perform repetitive functions after this point. The process steps may in some cases be recorded on a machine-readable medium.

[0136] For example, in some cases, a computer can be used to control imaging of the sample, e.g., using fluorescence microscopy, STORM, or other super-resolution techniques as described herein. In some cases, the computer can also control operations, such as drift correction in image analysis, physical positioning, hybridization and cluster alignment, cluster decoding (e.g., fluorescent cluster decoding), error detection or correction (e.g., as described herein), noise reduction, distinguishing foreground features from background features (such as noise or debris in the image), and the like. As an example, a computer can be used to control activation and / or excitation of signaling entities in a sample and / or acquisition of images of signaling entities. In one set of embodiments, the sample can be excited using light having various wavelengths and / or intensities, and a computer can be used to correlate the sequence of wavelengths of light used to excite the sample to the acquired images of the sample containing the signaling entities. For example, the computer can apply light having various wavelengths and / or intensities to the sample to obtain various 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, as described above, this information can be used to construct images, possibly at high resolution, and / or to determine the location of signaling entities. VI. Kit

[0137] The present disclosure provides a kit for carrying out the method described herein. A kit is provided for preparing FFPE tissue section samples and determining nucleic acid targets in the samples. In one embodiment, the kit includes the anchor probe and anchor agent disclosed herein with one or more other components. In one embodiment, the kit also includes the labeling agent disclosed herein for identifying cell type or tissue morphology. In some embodiments, the kit includes several different labeling agents that represent tissues 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) with one or more other components for MERFISH imaging.

[0138] One or more other kit components can include one or more buffers; nuclear counterstains; total RNA content counterstains; imaging buffers; software; and other components. The kit can also include instructions for using the kit components, as well as the use of any other reagents not included in the kit. The instructions can include variations that can be implemented.

[0139] In certain embodiments, kit components and protocols are provided herein for preparing FFPE tissue section samples for transcriptome analysis.In one embodiment, the kit comprises one or more of the following components: deparaffinization buffer, decrosslinking buffer, conditioning buffer, sample preparation washing buffer, formamide washing buffer, gel embedding premix, removal premix, gel cover slip, pre-anchor activator, anchor buffer and digestion premix.Anchor probe and target probe and their reagents for immobilizing target nucleic acid (e.g. RNA transcript) can also be provided, which are used to specifically detect target nucleic acid in prepared samples.In some embodiments, a kit is provided that comprises at least a first anchor agent and a second anchor agent or anchor probe.

[0140] Exemplary kit components are described in Example 1. Specific embodiments

[0141] The following embodiments of the methods described herein are provided.

[0142] Embodiment 1 is a method for detecting a nucleic acid target in a tissue sample, comprising: a. contacting a tissue sample containing a nucleic acid target with an anchor probe that specifically binds to the nucleic acid target; b. immobilizing a nucleic acid target binding anchor probe to at least a portion of the tissue sample within a gel; c. Removing the tissue sample within the polymer gel by removing or degrading non-targets; d. contacting the tissue sample with a plurality of nucleic acid probes capable of selectively binding nucleic acid targets; e. Detecting nucleic acid probes bound to nucleic acid targets within the tissue sample.

[0143] Embodiment 2 is a method for detecting a nucleic acid target in a tissue sample, comprising: a. contacting a tissue sample containing a nucleic acid target with an anchoring agent that includes a first chemical moiety that can react with and / or modify an internal base of the nucleic acid target and a second chemical moiety that can be incorporated into a polymer gel; b. immobilizing a nucleic acid target binding anchoring agent to at least a portion of the tissue sample within the gel; c. Removing the tissue sample within the polymer gel by removing or degrading non-targets; d. contacting the tissue sample with a plurality of nucleic acid probes capable of selectively binding nucleic acid targets; e. Detecting nucleic acid probes bound to nucleic acid targets within the tissue sample.

[0144] Embodiment 3 is a method for detecting a nucleic acid target in a tissue sample, comprising: a. contacting a tissue sample containing a nucleic acid target with an anchoring agent that includes a first chemical moiety that can react with and / or modify an internal base of the nucleic acid target and a second chemical moiety that can be incorporated into a polymer gel; b. contacting the tissue sample with an anchor probe that specifically binds a nucleic acid target; c. immobilizing a nucleic acid target binding anchor probe or a nucleic acid target binding anchor agent to at least a portion of the tissue sample within the gel; d. Removing the tissue sample within the polymer gel by removing or degrading non-targets; e. contacting the tissue sample with a plurality of nucleic acid probes capable of selectively binding nucleic acid targets; f. Detecting nucleic acid probes bound to nucleic acid targets within the tissue sample.

[0145] Example 4 is the method of any one of Examples 1 to 3, further comprising generating a codeword or barcode based on the distribution of bound nucleic acid probes within the sample.

[0146] Example 5 is the method of example 4, further comprising, for at least some of the codewords, matching the codeword with valid codewords in a codebook, and if no match is found, optionally applying error correction to the codeword to form a valid codeword or discarding the codeword.

[0147] Embodiment 6 is the method of any one of embodiments 1, 2, 4, and 5, wherein after steps (a) and (b), a removing step (c) is performed.

[0148] Embodiment 7 is the method of any one of embodiments 3 to 6, wherein after steps (a) to (c), a removing step (d) is performed.

[0149] Example 8 is the method of any one of Examples 1-7, wherein the steps are performed in the order recited.

[0150] Embodiment 9 is the method of any one of embodiments 1 to 8, wherein the tissue sample is a formalin-fixed paraffin-embedded (FFPE) tissue section.

[0151] Embodiment 10 is the method of any one of embodiments 1 to 8, wherein the tissue sample is deparaffinized and rehydrated prior to step (a).

[0152] Embodiment 11 is the method of any one of embodiments 1 to 10, further comprising deparaffinizing and rehydrating the tissue sample prior to step (a).

[0153] Embodiment 12 is the method of any one of embodiments 1-8, wherein the tissue sample is a fresh frozen tissue sample.

[0154] Embodiment 13 is the method of any one of embodiments 1 to 8, wherein the tissue sample is a fixed frozen tissue sample.

[0155] Embodiment 14 is the method of any one of embodiments 1 to 13, wherein the nucleic acid target is RNA.

[0156] Embodiment 15 is the method of any one of embodiments 1 to 13, wherein the nucleic acid target is DNA.

[0157] Embodiment 16 is the method of any one of embodiments 1-15, wherein the gel comprises polyacrylamide.

[0158] Embodiment 17 is the method of any one of embodiments 1 to 16, wherein at least some of the anchor probes include a poly dT moiety.

[0159] Embodiment 18 is the method of embodiment 17, wherein at least some of the anchor probes comprise alternating dT portions and locked dT portions.

[0160] Example 19 is the method of any one of Examples 16-18, wherein at least some of the anchor probes include an acrydite moiety capable of polymerizing with the gel.

[0161] Embodiment 20 is the method of embodiment 19, wherein an acrydite moiety is attached to the 5' end of the anchor probe.

[0162] Embodiment 21 is the method of embodiment 19, wherein an acrydite moiety is attached to the 3' end of the anchor probe.

[0163] Embodiment 22 is the method of embodiment 19, wherein an acrydite moiety is attached to an internal base of the anchor probe.

[0164] Embodiment 23 is the method of any one of embodiments 1 and 3-22, further comprising, prior to step (a), contacting the sample with an anchoring agent comprising a first chemical moiety capable of reacting with and / or modifying an internal base of the nucleic acid target and a second chemical moiety capable of being incorporated into a polymer gel.

[0165] Embodiment 24 is the method of embodiment 23, wherein the immobilizing step (b) further comprises polymerizing a gel within the tissue sample, wherein the second chemical moiety of the anchoring agent is copolymerized with the polymer gel.

[0166] Example 25 is the method of example 23 or 24, wherein at least some of the second chemical moieties of the anchor probes include an acrydite moiety capable of polymerizing with the gel.

[0167] Embodiment 26 is the method of any one of embodiments 23-25, wherein at least some of the nucleic acid targets are immobilized within the gel via both anchor probes and anchor agents bound to the nucleic acid targets.

[0168] Embodiment 27 is the method of any one of embodiments 1 to 26, wherein the non-target comprises a protein, a lipid, DNA, RNA, or an extracellular matrix.

[0169] Embodiment 28 is the method of any one of embodiments 1 to 27, wherein removing the tissue sample comprises removing or degrading proteins from the sample.

[0170] Embodiment 29 is the method of any one of embodiments 1 to 28, wherein removing the tissue sample comprises removing or degrading lipids from the sample.

[0171] Embodiment 30 is the method of any one of embodiments 1 to 29, wherein removing the tissue sample comprises removing or degrading non-target DNA from the sample.

[0172] Embodiment 31 is the method of any one of embodiments 1 to 30, wherein removing the tissue sample comprises removing or degrading extracellular matrix from the sample.

[0173] Embodiment 32 is the method of any one of embodiments 1 to 31, wherein removing the tissue sample comprises exposing the sample to an enzyme capable of degrading proteins.

[0174] Embodiment 33 is the method of any one of embodiments 1 to 32, wherein removing the tissue sample comprises exposing the sample to an enzyme capable of degrading DNA.

[0175] Embodiment 34 is the method of any one of embodiments 1 to 33, wherein removing the tissue sample comprises exposing the sample to an enzyme capable of degrading RNA.

[0176] Embodiment 35 is the method of any one of embodiments 1 to 34, wherein removing the tissue sample comprises exposing the sample to an enzyme capable of degrading the sugar or sugar-modified biomolecule.

[0177] Embodiment 36 is the method of any one of embodiments 1 to 35, wherein removing the tissue sample comprises exposing the sample to a detergent.

[0178] Embodiment 37 is the method of any one of embodiments 1 to 36, wherein removing the tissue sample comprises exposing the gel to a proteinase.

[0179] Embodiment 38 is the method of embodiment 37, wherein the proteinase comprises proteinase K.

[0180] Embodiment 39 is the method of any one of embodiments 1 to 38, wherein removing the tissue sample comprises exposing the gel to guanidine HCl.

[0181] Embodiment 40 is the method of any one of embodiments 1 to 39, wherein removing the tissue sample comprises exposing the gel to Triton X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether).

[0182] Embodiment 41 is the method of any one of embodiments 1 to 40, wherein removing the tissue sample comprises exposing the gel to sodium dodecyl sulfate.

[0183] Embodiment 42 is the method of any one of embodiments 1 to 41, wherein removing the tissue sample comprises exposing the gel to ethylenediaminetetraacetic acid.

[0184] Embodiment 43 is the method of any one of embodiments 1 to 42, wherein the plurality of nucleic acid probes comprises smFISH probes.

[0185] Embodiment 44 is the method of any one of embodiments 1 to 43, wherein the plurality of nucleic acid probes comprises MERFISH probes.

[0186] Embodiment 45 is the method of any one of embodiments 1 to 44, wherein step (d) further comprises amplifying the nucleic acid probe.

[0187] Embodiment 46 is the method of any one of embodiments 1 to 45, wherein detecting comprises imaging using optical microscopy.

[0188] Embodiment 47 is the method of any one of embodiments 1 to 46, wherein detecting comprises imaging using fluorescence microscopy.

[0189] Embodiment 48 is the method of embodiment 47, comprising imaging using epifluorescence microscopy, total internal reflection microscopy, highly inclined thin-illumination (HILO) microscopy, light sheet microscopy, scanning confocal microscopy, scanning line confocal microscopy, spinning disk confocal microscopy, or other equivalent conventional microscopy techniques.

[0190] Embodiment 49 is the method of embodiment 47, comprising imaging using multiplex fluorescent in situ hybridization.

[0191] Embodiment 50 is the method of embodiment 47, comprising imaging using multiplex error-robust fluorescence in situ hybridization (MERFISH).

[0192] Embodiment 51 is the method of any one of embodiments 47 to 50, comprising imaging using multiple rounds of fluorescent in situ hybridization.

[0193] Embodiment 52 is the method of any one of embodiments 1 to 51, wherein the nucleic acid probe comprises a targeting sequence and one or more lead sequences.

[0194]

[0036] Embodiment 53 is the method of embodiment 52, further comprising determining the lead sequence based on determining binding of the lead sequence bound to the target nucleic acid target.

[0195] Embodiment 54 is the method of embodiment 53, wherein the codeword or barcode is generated based on determining the read sequence in a gel.

[0196] Embodiment 55 is the method of any one of embodiments 52-54, wherein the lead sequences are obtained from a set of orthogonal sequences that have less than 15 base pairs of homology with each other and with the nucleic acid species in the sample.

[0197] Embodiment 56 is a method of any one of embodiments 4 to 55, wherein each of the code words represents one of a plurality of different nucleic acid targets and includes a plurality of binary values ​​of 1 and 0, where a value of 1 is obtained when a signal is detected at the respective location within the sample and a value of 0 is obtained when no signal is detected.

[0198] Embodiment 57 is the method of any one of embodiments 4 to 56, wherein a codeword is generated that represents a plurality of different nucleic acid targets at locations within the sample, each of the codewords representing one of the plurality of different nucleic acid targets and including a plurality of binary values ​​of 1 and 0, wherein a value of 1 is obtained when a signal is detected from one of the plurality of readout probe hybridization complexes or one of the plurality of different readout probe hybridization complexes at the respective locations within the sample, and a value of 0 is obtained when a signal is not detected from one of the plurality of readout probe hybridization complexes or one of the plurality of different readout probe hybridization complexes at the respective locations within the sample.

[0199] Embodiment 58 is the method of any one of embodiments 5 to 57, wherein the codebook includes valid codewords for a plurality of nucleic acid targets.

[0200] Embodiment 59 is the method of any one of embodiments 5 to 58, wherein step (h) of matching the codeword with a valid codeword in the codebook includes comparing the codeword with the valid codewords in the codebook, and if the codeword does not match one of the valid codewords in the codebook, applying an error detection or correction system, matching the codeword with another of the valid codewords in the codebook, or discarding the codeword, wherein the codebook includes valid codewords for a plurality of nucleic acid targets.

[0201] Embodiment 60 is the method of any one of embodiments 1 to 59, wherein prior to step (a), the tissue sample is contacted with at least one labeling agent for labeling at least one cellular component.

[0202] Embodiment 61 is the method of any one of embodiments 1 to 60, further comprising, prior to step (a), contacting the tissue sample with at least one labeling reagent for labeling at least one cellular component prior to step (a).

[0203] Embodiment 62 is the method of embodiment 60 or 61, wherein at least one labeling reagent comprises at least three oligonucleotide-conjugated labeling probes, each comprising an anchor portion capable of being attached to a gel and (2) an oligonucleotide conjugated to a binding portion capable of being bound to a cellular component, the binding portion comprising: (i) a protein-binding portion capable of being bound to a cellular protein component; (ii) a carbohydrate-binding portion capable of being bound to a cellular carbohydrate component; or (iii) a chemical binding portion capable of being bound to or incorporated into the cellular component.

[0204] In some preferred embodiments, the present disclosure provides a method for anchoring a target nucleic acid in a matrix and removing non-target cellular components, the method comprising: contacting a formalin-fixed paraffin-embedded (FFPE) tissue sample with at least two anchoring agents, the first anchoring agent forming a covalent bond with the target nucleic acid and the second anchoring agent comprising an oligonucleotide that hybridizes with the target nucleic acid; embedding the sample in a polymer matrix, the first and second anchoring agents each forming a covalent bond with the polymer matrix; and removing the non-target cellular components from the polymer matrix, leaving the target nucleic acid anchored in the polymer matrix to form a matrix-anchored target nucleic acid sample. In some preferred embodiments, the first anchoring agent is an alkylating agent. In some preferred 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 some preferred embodiments, the second anchoring agent comprises alternating dT and locked dT moieties that hybridize to the target nucleic acid. In some preferred embodiments, the second anchoring agent comprises a poly dT moiety that hybridizes to the target nucleic acid. In some preferred embodiments, the first anchoring agent and the second anchoring agent each comprise an acrydite moiety that covalently binds to the polymer matrix. In some preferred embodiments, the first anchoring agent is an alkylating agent derivatized with an acrydite moiety. In some preferred embodiments, the second anchoring agent comprises a poly dT moiety that hybridizes to the target nucleic acid and an acrydite moiety that covalently binds to the polymer matrix. In some preferred embodiments, the target nucleic acid is RNA. In some preferred embodiments, the target nucleic acid is DNA. In some preferred embodiments, the method further comprises contacting the anchored target nucleic acid sample with one or more primary oligonucleotide probes that hybridize to the target nucleic acid.In some preferred embodiments, the primary oligonucleotide probe is a single molecule (sm) FISH probe or a multiplex error-robust fluorescent in situ hybridization (MERFISH) probe. In some preferred embodiments, the one or more primary oligonucleotide probes include a first portion including a target sequence and a second portion including one or more lead sequences. In some preferred embodiments, the method further includes determining a lead sequence based on contacting the one or more primary oligonucleotide probes with a plurality of secondary nucleic acid probes including recognition sequences that hybridize to the lead sequence of the primary nucleic acid probe. In some preferred embodiments, the method further includes imaging using multiplex fluorescent in situ hybridization, including contacting the anchored target nucleic acid sample with one or more primary oligonucleotide probes that hybridize to the target nucleic acid, and including one or more successive steps of adding a plurality of secondary nucleic acid probes that include a label moiety. In some preferred embodiments, the method further includes imaging using a multiplex error-robust fluorescent in situ hybridization (MERFISH) probe, including contacting the anchored target nucleic acid sample with one or more MERFISH probes that hybridize to the target nucleic acid. In some preferred embodiments, the methods include imaging using multiple rounds of fluorescent in situ hybridization, in each round using one or more different secondary nucleic acid probes, each conjugated to a spectrally distinct fluorescent label, to simultaneously read out multiple readout sequences.

[0205] In some preferred embodiments, the present disclosure provides a method of anchoring a target RNA in a matrix and removing non-target cellular components, the method comprising: contacting a formalin-fixed, paraffin-embedded (FFPE) tissue sample with at least two anchoring agents, where a first anchoring agent comprises an alkylating agent that forms a covalent bond with the target nucleic acid, and a second anchoring agent comprises a poly-T sequence that is complementary to and hybridizes with the target RNA, and where the first anchoring agent and the second anchoring agent each comprise an acrydite moiety that is covalently attached to the matrix; embedding the sample in a polymer matrix, where the first and second anchoring agents each form a covalent bond with the polymer matrix; and removing the non-target cellular components from the polymer matrix, where the target RNA remains anchored to the polymer matrix to form a matrix-anchored target RNA sample. In some preferred 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 some preferred embodiments, the second anchoring agent comprises alternating dT moieties that hybridize to the target RNA and locked dT moieties. In some preferred embodiments, the method further comprises contacting the anchored target nucleic acid sample with one or more primary oligonucleotide probes that hybridize to the target nucleic acid. In some preferred embodiments, the primary oligonucleotide probes are single molecule (sm) FISH probes or multiplex error-robust fluorescent in situ hybridization (MERFISH) probes. In some preferred embodiments, the one or more primary oligonucleotide probes comprise a first portion that includes a target sequence and a second portion that includes one or more lead sequences. In some preferred embodiments, the method further includes determining a lead sequence based on contacting one or more primary oligonucleotide probes with a plurality of secondary nucleic acid probes that comprise recognition sequences that hybridize to the lead sequence of the primary nucleic acid probe.In some preferred embodiments, the method further comprises imaging using multiplex fluorescent in situ hybridization, comprising contacting the anchored target nucleic acid sample with one or more primary oligonucleotide probes hybridized to the target nucleic acid, and comprising one or more successive steps of adding a plurality of secondary nucleic acid probes comprising a label moiety. In some preferred embodiments, the method further comprises imaging using multiplex error-robust fluorescent in situ hybridization (MERFISH) probes, comprising contacting the anchored target nucleic acid sample with one or more MERFISH probes hybridized to the target nucleic acid. In some preferred embodiments, the method further comprises imaging using multiple rounds of fluorescent in situ hybridization, in which multiple readout sequences are simultaneously read out in each round using one or more different secondary nucleic acid probes, each conjugated to a spectrally distinct fluorescent label.

[0206] In some preferred embodiments, the present disclosure provides a method for imaging a target nucleic acid in a matrix and removing non-target cellular components, the method includes contacting a formalin-fixed paraffin-embedded (FFPE) tissue sample with at least two anchoring agents, the first anchoring agent forming a covalent bond with the target nucleic acid and the second anchoring agent comprising an oligonucleotide hybridizing with the target nucleic acid; embedding the sample in a polymer matrix, the first and second anchoring agents each forming a covalent bond with the polymer matrix; removing the non-target cellular components from the polymer matrix, leaving the target nucleic acid anchored in the polymer matrix to form a matrix-anchored target nucleic acid sample; and contacting the anchored target nucleic acid sample with one or more primary oligonucleotide probes hybridizing to the target nucleic acid, and a plurality of secondary nucleic acid probes comprising a fluorescent label and a recognition sequence hybridizing to the sequence of the primary nucleic acid probe, and imaging the target nucleic acid. In some preferred embodiments, the first anchoring agent is an alkylating agent. In some preferred 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 some preferred embodiments, the second anchoring agent comprises alternating dT and locked dT moieties that hybridize to the target nucleic acid. In some preferred embodiments, the second anchoring agent comprises a poly dT moiety that hybridizes to the target nucleic acid. In some preferred embodiments, the first anchoring agent and the second anchoring agent each comprise an acrydite moiety that covalently binds to the polymer matrix. In some preferred embodiments, the first anchoring agent is an alkylating agent derivatized with an acrydite moiety. In some preferred embodiments, the second anchoring agent comprises a poly dT moiety that hybridizes to the target nucleic acid and an acrydite moiety that covalently binds to the polymer matrix. In some preferred embodiments, the target nucleic acid is RNA.In some preferred embodiments, the target nucleic acid is DNA. In some preferred embodiments, the primary oligonucleotide probe is a single molecule (sm) FISH probe or a multiplex error-robust fluorescent in situ hybridization (MERFISH) probe. In some preferred embodiments, the one or more primary oligonucleotide probes include a first portion including a target sequence and a second portion including one or more lead sequences. In some preferred embodiments, the method further includes determining a lead sequence based on contacting the one or more primary oligonucleotide probes with a plurality of secondary nucleic acid probes including recognition sequences that hybridize to the lead sequences of the primary nucleic acid probes. In some preferred embodiments, the secondary nucleic acid probes are added in one or more successive steps, and imaging is performed between each successive round of adding the secondary nucleic acid probes. In some preferred embodiments, the method further includes imaging using a multiplex error-robust fluorescent in situ hybridization (MERFISH) probe, and includes contacting the anchored target nucleic acid sample with one or more MERFISH probes that hybridize to the target nucleic acid. In some preferred embodiments, the method further comprises imaging using multiple rounds of fluorescent in situ hybridization, in each round using one or more different secondary nucleic acid probes, each conjugated to a spectrally distinct fluorescent label, to simultaneously read out multiple readout sequences.

[0207] In some preferred embodiments, the present disclosure provides a method for imaging a target RNA in a matrix and removing non-target cellular components, the method comprising: contacting a formalin-fixed paraffin-embedded (FFPE) tissue sample with at least two anchoring agents, where a first anchoring agent comprises an alkylating agent that forms a covalent bond with the target nucleic acid, and a second anchoring agent comprises a poly-T sequence that is complementary to and hybridizes with the target RNA, and the first anchoring agent and the second anchoring agent each comprise an acrydite moiety that is covalently attached to the matrix; embedding the sample in a polymer matrix, where the first and second anchoring agents each form a covalent bond with the polymer matrix; removing the non-target cellular components from the polymer matrix, where the target RNA remains anchored in the polymer matrix to form a matrix-anchored target RNA sample; and contacting the anchored target RNA sample with one or more primary oligonucleotide probes that hybridize to the target RNA, and a plurality of secondary nucleic acid probes that comprise fluorescent labels and recognition sequences that hybridize to sequences of the primary nucleic acid probes, and imaging the target nucleic acid. In some preferred 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 some preferred embodiments, the second anchoring agent comprises alternating dT portions hybridizing to the target nucleic acid and locked dT portions. In some preferred embodiments, the primary oligonucleotide probe is a single molecule (sm) FISH probe or a multiplex error-robust fluorescent in situ hybridization (MERFISH) probe. In some preferred embodiments, the one or more primary oligonucleotide probes comprise a first portion comprising a target sequence and a second portion comprising one or more lead sequences.In some preferred embodiments, the method further comprises determining a lead sequence based on contacting one or more primary oligonucleotide probes with a plurality of secondary nucleic acid probes comprising recognition sequences that hybridize to the lead sequence of the primary nucleic acid probe. In some preferred embodiments, the secondary nucleic acid probes are added in one or more successive steps, and imaging is performed between each successive round of adding the secondary nucleic acid probes. In some preferred embodiments, the method further comprises imaging using a multiplex error-robust fluorescent in situ hybridization (MERFISH) probe, comprising contacting the anchored target nucleic acid sample with one or more MERFISH probes that hybridize to the target nucleic acid. In some preferred embodiments, the method further comprises imaging using multiple rounds of fluorescent in situ hybridization, in each round, one or more different secondary nucleic acid probes, each conjugated to a spectrally distinct fluorescent label, are used to simultaneously read out a plurality of readout sequences.

[0208] Although an embodiment of the invention will be described in detail, it will be understood that other embodiments are contemplated. Accordingly, it is not intended that the invention be limited in scope to the details of construction and the 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 used for the sake of clarity.

[0209] It should also be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to a "probe" is intended to include multiple probes, reference to a "target" is intended to include multiple targets, and so forth.

[0210] The term "or" is used herein in its inclusive sense, ie, equivalent to "and / or," unless the context clearly dictates otherwise.

[0211] Numerical ranges include the numbers that define the range. Measurements and measurable values ​​are understood to be approximations taking into account significant digits and errors associated with measurement. Thus, unless indicated to the contrary, the numerical parameters set forth in the following description and appended claims are approximations that may vary depending on 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.

[0212] The term "about" or "approximately" refers to 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 on the degree of variation that does not substantially affect the properties of the described subject matter.

[0213] 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 specified compounds, elements, particles, or method steps are present in a composition or article or method, but does not exclude the presence of other compounds, materials, particles, or method steps even if they have the same function as the one specified.

[0214] Also, in describing the embodiments, terminology is used for the sake of clarity: each term is intended to be accorded its broadest meaning as understood by one of ordinary skill in the art and to encompass all technical equivalents that operate in a similar manner to accomplish a similar purpose.

[0215] It should also be understood that the reference to one or more method steps does not preclude the presence of additional or intervening method steps between those explicitly identified steps. Similarly, it should also be understood that the reference to one or more components in a structure or system does not preclude the presence of additional or intervening components between those explicitly identified components.

[0216] Those skilled in the art will appreciate that the above-mentioned figures and following examples are for illustrative purposes only, and neither the figures nor the examples are intended to limit the scope of the disclosed teachings in any way. VII. Examples

[0217] The following examples are provided to illustrate certain disclosed embodiments and should not be construed in any way as limiting the scope of the disclosure. [Example 1] In situ single-cell transcriptome imaging by MERFISH in FFPE samples ("Protocol A")

[0218] This example shows a sample protocol ("Protocol A") for preparing FFPE samples for MERFISH imaging. It is understood that this Protocol A can be used to anchor fragmented RNA with any downstream in situ imaging, not just MERFISH imaging, and this example is provided for illustrative purposes only and does not imply any limitations on the methods described herein. Protocol A and a comparative protocol ("Protocol B") (adding MERFISH and another anchoring agent to the sample before removing FFPE and fresh frozen samples) are shown in Figure 1.

[0219] In certain embodiments, Protocol A can be performed as described below. 1) Fiducial coating on a slide (e.g., MERSCOPE Slide, Vizgen, #20400001) that allows image registration for multiple rounds of imaging: a. Make a bead solution (e.g., Spherotech fluorescent beads, catalog number: FP-0252-2) at a 1:500 dilution in PBS. b. Add 500 μl of bead solution to the center of the bead-precoated coverslip, covering approximately 80% of the total area of ​​the coverslip. Avoid allowing the bead solution to flow under the coverslip. Incubate for 10 minutes at room temperature. c. Aspirate the bead solution. d. Wash once with 500 μl PBS and aspirate the PBS to prevent the bead solution from running down the coverslip. e. Add an additional 500 μl of PBS to the center of the coverslip. 2) Sectioning FFPE slices onto standard coated coverslips a. Immediately prior to sectioning the FFPE slices, aspirate the PBS solution. b. Place one or more FFPE slices, 4-5 µm thick, onto the prepared coverslip. c. Allow the coverslips to dry at room temperature for 2-3 min. d. Dry the coverslips in an oven at 50-55 °C for 10 min. e. Thoroughly remove all remaining water droplets. f.FFPE coverslips can be used immediately or stored in a -20 degree freezer for 1-2 months. 3) Deparaffinization and rehydration of FFPE slices Add 400 μl of deparaffinization solution (e.g., Zymo Research, D3067-1-20) to the center of the coverslip. Allow the solution to gradually cover the entire coverslip. b. Place the coverslip in a 50-55°C oven for 5 minutes. c. Aspirate the solution. d. Add an additional 350ul of deparaffinization solution to the center of the coverslip, making sure the solution covers the entire coverslip. e. Incubate at room temperature for 2 minutes. f. Aspirate the solution. g. Rinse the dish containing the coverslip with 5 ml of 100% ethanol and aspirate. h. Add 5 ml of 100% ethanol to the dish containing the coverslips, incubate at room temperature for 2 minutes, and aspirate. i. Repeat step 3(h) one more time. j. Add 5 ml of 90% ethanol to the dish containing the coverslips, incubate at room temperature for 5 minutes, and aspirate. k. Add 5 ml of 70% ethanol to the dish containing the coverslips, incubate at room temperature for 5 minutes, and aspirate. l. Coverslips can be stored in 70% ethanol for up to 1 month. 4) Antigen retrieval of FFPE slices Add 5 ml of citrate-based antigen retrieval buffer to rinse and aspirate. Add 5 ml of citrate-base antigen retrieval buffer. Place the dish in a 90-95 degree oven for 15 minutes. c. Leave the dish on the bench at room temperature for 5 minutes and aspirate. 5) Cell segmentation and immunostaining of protein biomarkers for protein imaging. In this step, cell morphology and protein staining are performed. a. Add 5 ml of PBS to the dish to rinse and aspirate. b. Add 100 μl of blocking solution containing 3% bovine serum albumin (BSA) to cover the entire tissue. Incubate at room temperature for 45-60 minutes. Aspirate. c.Add 100 μl of blocking solution containing 3% BSA + primary antibody for cell boundary 3 or other antibodies diluted according to the given concentration. The solution should cover the entire tissue. Incubate for 60 minutes at room temperature. d. Add 5ml of PBS to the dish. Incubate on bench for 5 minutes. Shake and aspirate occasionally. e. Repeat step 5(d) once. f. Add 100 μl of blocking solution containing 3% BSA + secondary antibody for cell border / cell morphology staining according to the given concentration. Incubate at room temperature for 60 minutes. g. Add 5 ml of PBS to the dish. Incubate on bench for 5 minutes. Shake and aspirate occasionally. h. Repeat step 5(g) twice. 6) Pre-anchoring of FFPE slices to prime the tissue for anchoring: In this step, a first anchoring agent is added to the sample to form covalent bonds with nucleic acids. a. Add 4 ml of Anchor Pretreatment Dilution Buffer to the dish, rinse and aspirate. b. Add 4 ml of Anchor Pretreatment Dilution Buffer to the dish. Incubate in a 37 degree incubator for 30 minutes and aspirate. c. Add 100 μl of Anchor Pretreatment Solution to the tissue to completely cover it. Incubate in a 37°C incubator for 2 hours. 7) Anchoring of RNA molecules with polyT probes: In this step, a second anchoring agent or anchor probe is added to the sample and hybridizes with the polyA tails of the mRNA present in the sample. a. Add 5ml sample prep wash buffer to the dish to rinse and aspirate. b. Add 5 ml of formamide wash buffer to the dish. Incubate in a 37 degree incubator for 30 minutes. Aspirate the solution. c. Add 75 μl of PolyT anchor buffer to the center of the tissue. Use a 2×2 cm piece of parafilm to spread and cover the entire tissue. Incubate in a 37° incubator overnight (at least 16 hours). d. Add 5 ml of formamide wash buffer to the dish. Incubate in a 47 degree incubator for 15 minutes and aspirate. 8) The gel is embedded and the tissue is removed to remove the autofluorescence background. In this step, when the first and second anchoring agents form covalent bonds with the polyacrylamide gel, respectively, the mRNA is immobilized in the gel and the non-immobilized cellular components are removed, resulting in a gel-immobilized target nucleic acid sample. Add 5 ml of sample prep wash buffer to rinse dish. Aspirate. b. Gel coverslips (Vizgen, Inc) are cleaned by spraying with RnaseZap solution and wiping with a Kimwipe, followed by spraying with 70% ethanol and wiping with a Kimwipe. c. Add 50 μL of Gel Slick Solution (VWR, Catalog No. 12001-812) to the gel coverslip and gently wipe with a Kimwipe to spread the Gel Slick. d. Prepare a gel embedding solution containing polyacrylamide, 0% w / v ammonium persulfate solution, and N,N,N',N'-tetramethylethylenediamine. e. Aspirate the sample prep wash buffer. Retain 100 µL of gel embedding solution in a small tube. Add the remaining 5 mL of embedding solution and incubate at room temperature for 1 minute. f. Using a pipette, transfer most of the embedding solution to a waste tube (to monitor gel formation). g. Dry the slides by aspirating, leaving enough liquid to cover the tissue sections. h. Add 50 μL of the retained gel embedding solution to the tissue section. i. Place the tips of a pair of tweezers on the area of ​​the MERSCOPE slide, without touching the tissue section. Use the tweezers to pick up a 20 mm Gel Slick treated gel coverslip. With the Gel Slick treated side facing down towards the tissue, place the edge of the gel coverslip against the tweezers tips placed on the MERSCOPE slide to create stability and slowly lower the gel coverslip onto the tissue section to spread the gel embedding solution. Adjust if necessary so that the gel coverslip is centered on the MERSCOPE slide. Gently press the gel coverslip to squeeze out excess gel embedding solution and remove excess gel embedding solution by suction. j. Incubate at room temperature for 1.5 hours. k. Be sure to wear eye protection during this step. Gently lift off the 20 mm Gel Slick treated gel coverslip with the sharp end of a hobby blade and properly discard the gel coverslip. l. Warm the depletion premix (containing SDS, 2xSSC and Trion-x 100) at 37°C for 30 min before use. The depletion premix should be a clear solution before use. If the solution is cloudy, warm it until the solution becomes clear. Prepare the depletion solution containing the depletion premix and proteinase K (volume ratio 100:1). m. (Optional) In case of resistant tissue, add 200 μl of digestion premix containing a mixture of enzymes capable of digesting 200 μl of connective tissue + 10 μl of Rnase inhibitor on top of the gel. Incubate in a 37 degree incubator for 1-6 hours. Add n.5 ml of stripping solution to the dish. Seal the dish with a piece of parafilm. Incubate in a 47 degree incubator for 18-24 hours. After 18 hours of incubation, monitor the clarity of the tissue. Once the tissue is clear, proceed to the next step. o. Photobleach for 3-4 hours. (Note: this photobleaching step can be performed after "step 3" above) p. Samples can be stored in 5 ml of stripping solution in a 37 degree incubator for up to 1 week. 9) For imaging of the anchored mRNA, hybridization with MERFISH-coded probes ("primary oligonucleotide probes") designed to hybridize to multiple target nucleic acids within the anchored mRNA. Add 5 ml of sample prep wash buffer. Shake for 5 minutes at room temperature on a shaker and aspirate. b. Repeat step 9(a) three times. c. Add 5 ml of formamide wash buffer to the dish. Incubate in a 37 degree incubator for 30 minutes. Aspirate the solution. d. Add 80 μl of MERFISH coded probe ("primary oligonucleotide probe") onto the gel where the mRNA was anchored. Use a 2 x 2 cm piece of parafilm to spread it out and cover the entire tissue. Incubate in a 37° incubator for 2 days (36-48 hours). 10) Wash to remove excess or unbound primary oligonucleotide probe: Add 5 ml of formamide wash buffer to the dish. Incubate in a 47 degree incubator for 30 minutes and aspirate. b. Repeat 10(a) once. c. Samples can be stored in 5ml of stripping solution in a 37 degree incubator for up to 1 week. After storage, samples should be washed with 5ml of sample prep wash buffer. Shake for 5 minutes at room temperature on a shaker and aspirate. Repeat wash once with 5ml of sample prep wash buffer. 11) (Possibly) Increase (amplify) the signal Add 100ul of enhancer solution to the gel containing the tissue. Incubate in a 37 degree incubator overnight (>16 hours). b. Add 5ml of formamide wash buffer to the dish. Incubate on bench at room temperature for 5 minutes. c. Add 5ml of formamide wash buffer to the dish. Incubate on bench at room temperature for 10 minutes. d. Rinse with 5 ml of sample prep wash buffer. 12) Data acquisition and analysis Add 3 mL of DAPI and Poly-T staining reagent and incubate on rocker for 15 min. b. Wash with 5 mL of formamide wash buffer and incubate for 10 min. c. Wash with 5 ml of sample prep wash buffer. d. The sample coverslip is placed onto the MERSCOPE (Vizgen, Inc.) and secondary nucleic acid probes ("readout probes") are added sequentially to acquire data and then analyzed. [Example 2] In situ single-cell transcriptome imaging by MERFISH in FFPE samples

[0220] This example shows the result of MERFISH imaging in FFPE samples after target fixation and removal method according to the present disclosure.Following the fixation of target nucleic acid and removal of non-target cellular components, a primary nucleic acid probe designed to hybridize with the fixed target nucleic acid is added, and then a readout probe that binds to the complementary sequence ("read sequence") of the primary nucleic acid probe and contains a fluorescent label is added.More details of the method are provided above in Example 1.

[0221] Figure 2A-C show MERFISH imaging using a 128-plex gene panel in FFPE mouse small intestine. Figure 2A shows the spatial distribution of selected genes throughout the tissue. Figure 2B shows the tissue morphology visualized by selected transcripts (left) and the distribution of all transcripts in a magnified area.

[0222] To determine the accuracy of these measurements, we compared copy numbers per gene determined by MERFISH for tissue sections ("MERFISH counts" in Figure 2C, "MERFISH") to abundances determined by RNA-seq from the same tissue samples as measured by FPKM. As shown in Figure 2C, MERFISH counts correlated strongly with bulk RNA sequencing FPKM data (r = 0.81), indicating that the measurements are quantitative and highly accurate.

[0223] Similarly, Figure 3 shows MERFISH imaging using a 244-plex gene panel in FFPE human colon cancer. Figure 3A shows the spatial distribution of selected genes across the tissue. Figure 3B shows tissue morphology visualized by selected transcripts (left) and the distribution of all transcripts in a magnified area (right). As shown in Figure 3C, MERFISH counts are strongly corrected (r=0.80) with bulk RNA sequencing FPKM data, indicating that the measurement is quantitative and highly accurate.

[0224] Figure 4 also shows MERFISH imaging using a 483-plex gene panel in FFPE mouse brain. Figure 4A shows the spatial distribution of selected genes throughout the tissue. Figure 4B shows the tissue morphology visualized by selected transcripts (left) and the distribution of all transcripts in a magnified area (right). As shown in Figure 4C, MERFISH counts are strongly corrected (r = 0.88) with bulk RNA sequencing FPKM data, indicating that the measurement is quantitative and highly accurate. Example 3: Comparison with other imprinting and tissue removal protocols

[0225] This example provides a comparison of sample preparation according to certain embodiments of the present disclosure ("Protocol A") with a comparative protocol ("Protocol B") in which MERFISH (coded probe or primary oligonucleotide probe) and anchor probe are added to the sample prior to clearing FFPE and fresh frozen samples (Figure 1). FFPE mouse small intestine samples were processed with Protocol A or Protocol B. Fresh frozen mouse small intestine samples were also processed with Protocol B. All samples were then imaged with the MERSCOPE protocol (i.e., MERFISH coded probe and readout probe were added).

[0226] Figure 5 shows the average counts of transcripts per field of view (FOV) for both conditions, indicating that samples prepared according to protocol A showed higher levels of transcript detection, indicating that more nucleic acid in the sample was anchored to the matrix and available for hybridization to the coded probe. [Example 4] MERFISH imaging in human FFPE tissue samples

[0227] This example shows MERFISH measurements of human FFPE tissue samples prepared according to Protocol A of the present disclosure.

[0228] Figure 6 shows MERFISH imaging with a 244-plex gene panel in 15 different archival human FFPE samples. For each dataset, 1000-2000 fields of view were captured, generating millions to hundreds of millions of counts per tissue slice. The top plot in Figure 6A shows the average counts per field of view at an area size of 200 x 200 µm, demonstrating that the workflow works robustly across a wide range of FFPE samples obtained from normal human tissues (top) and human tumors (bottom). Figure 6B shows that the quality of the MERFISH data correlates with the quality of the RNA of the samples, as indicated by the DV200 values. DV200 is the percent of RNA fragments greater than 200 nucleotides in the sample. As shown in Figure 6C, MERFISH counts were robustly corrected (r=0.88) with bulk RNA sequencing FPKM data across diverse human tissue samples. [Example 5] Comparison of various human FFPE samples

[0229] This example provides a comparison of MEFISH imaging of human FFPE samples according to certain embodiments of the present disclosure ("Protocol A") with a comparative protocol ("Protocol B") that can be used for interrogation of fresh frozen samples as described in Example 3.

[0230] Figure 7 shows that Protocol A provides higher sensitivity and precision than the comparative protocol (Protocol B) across a variety of sample types. The average MERFISH counts per field of view with an area size of 200 x 200 μm were shown for samples across a variety of tissue types (mouse small intestine, mouse brain, human kidney and human colon cancer) prepared by Protocol A and Protocol B, with MERFISH counts including high correlation. [Example 6] Single cell analysis in FFPE human melanoma samples

[0231] This example demonstrates single cell analysis in FFPE human melanoma samples. Cell segmentation was performed after antibody-based cell border staining as described in Example 1 above. Uniform Manifold Approximation and Projection (UMAP) clustering, a dimensionality reduction method that captures the variability in a limited number of random variables and facilitates the visualization of data sets with tens to thousands of dimensions, was used to define and identify cell types in mixed populations based on gene expression profiles of individual cells.

[0232] The results showed that the spatial distribution of selected cell types throughout the tissue and the spatial distribution of selected gene transcripts within selected cell types in melanoma were obtained. This indicates that MERFISH-mediated spatial transcriptome technology can be performed with high detection efficiency and single molecule resolution in FFPE samples when prepared according to the method described in Example 1, ultimately providing gene expression profiles in tissue samples in situ. Data not shown. [Example 7] Single cell analysis of various FFPE samples

[0233] This example demonstrates single cell analysis in various FFPE samples following the MERFISH protocol (Protocol A) according to the method described in Example 1: A) Mouse small intestine. B) Mouse brain. C) Human liver cancer. D) Human kidney. E) Human lung. F) Human ovarian cancer. G) Human uterine cancer. H) Human lung cancer. (Top) UMAP clustering for cell type identification; (Bottom) Spatial distribution of identified cell types.

[0234] The results show that it was possible to perform in situ single-cell transcriptome imaging of selected gene transcripts in various tissue samples from human and mouse when FFPE samples were prepared according to the methods described in Example 1. Data not shown. Example 8: Immuno-oncology data generated using FFPE samples

[0235] A summary of the data generated using FFPE for eight sample types, 16 datasets, 500 genes, approximately 4 billion transcripts and approximately 9 million cells is shown in Table 1 below. [Table 1]

[0236] Figures 10A-C show that the FFPE workflow is highly sensitive, accurate and reproducible. Figure 10A shows the correlation of MERSCOPE data between two human ovarian cancer slices from the same patient. The correlation coefficient is 0.99, indicating high reproducibility of the measurements. In Figure 10B, human ovarian cancer sample 1 was analyzed by MERSCOPE with a 500-gene panel and an adjacent slice was analyzed by bulk RNA sequencing. The correlation analysis between MERFISH counts and FPKM values ​​from bulk RNA sequencing is shown. The correlation coefficient is 0.82, indicating high precision of the measurements. Figure 10C presents the correlation analysis between MERSCOPE data and bulk RNA sequencing performed across 14 cancer samples, where the correlation coefficient indicates high precision across multiple cancer types and replicates.

[0237] Figures 11A-11F show that the FFPE cell segmentation workflow enables true atlasing in dense tissues. In Figure 11A, FFPE human liver cancer was immunostained with a cell boundary staining kit and DAPI for nuclear staining. Figure 11B shows cells segmented using a deep learning based cell segmentation algorithm. Polygon masks of each identified cell are shown. Figure 11C shows a UMAP visualization of the 17 different cell types identified in human liver cancer, generated from MERFISH transcript data. Figure 11D shows the spatial distribution of the identified cell types across the tissue within the boxed area of ​​Figure 11B. Figure 11E shows the spatial distribution of fibroblasts in the boxed area of ​​Figure 11B. Fibroblast marker gene COL1A1. Figure 11F shows the partial distribution of endothelial cells in the boxed area of ​​B. Endothelial marker gene PECAM1.

[0238] Figure 12 shows the spatial distribution of identified cell types across different FFPE tumor samples. Different cancer samples including breast, colon, melanoma, lung, liver, ovarian, prostate and uterine cancer were analyzed by MERSCOPE using a 500 gene panel with a cell border staining kit to label cell borders. Cells were segmented and subjected to single cell analysis. Identified cells in each sample were colored to show the spatial distribution of different cells across the samples. Scale bar: 1 mm.

[0239] FIG. 13 shows that the FFPE protocol can be used to show the spatial distribution of expression of select genes (ACTA2, CD3D, LGR5, MK167 and PECAM1) in human breast cancer. FIG. 13A shows the spatial distribution of select genes including ACTA2 (green), CD3D (red), LGR5 (light green), MKI67 (magenta) and PECAM1 (blue) from 500 genes analyzed across the tissue. Scale bar: 1 mm. FIG. 13B provides a magnification of the boxed area in FIG. 13A. Scale bar: 1 mm. FIG. 13C shows a magnification of the boxed area in FIG. 13B, showing the cell boundary polygon mask in grey. Scale bar: 250 mm.

[0240] Figures 14A-E show that FFPE protocols can be used to identify and map cell types in human breast cancer. Figure 14A provides a UMAP visualization of the different cell types identified in human breast cancer, generated from MERFISH transcript data. Figure 14B shows the spatial distribution of the 14 identified cell types across the tissue. Figure 14C shows the spatial distribution of the identified cell types within the boxed area of ​​Figure 14B. Figure 14D shows the spatial distribution of two types of fibroblasts (fibroblast 1 in green and fibroblast 2 in red) within the boxed area of ​​Figure 14C. Both types of fibroblasts express the COL1A1 gene, while fibroblast 2 expresses the proliferation marker MKI67. Figure 14E provides a dot plot showing the marker genes for each cell type.

[0241] FIG. 15 shows that FFPE protocols can be used to characterize immune cell types in the tumor microenvironment. FIG. 15A shows that T / NK cell clusters from breast cancer samples were selected for subclustering analysis. UMAP visualization of the subclustering analysis shows seven distinct immune cell subtypes within human breast cancer. FIG. 15B provides dot plots showing marker genes for each immune cell type, including myeloid cells, CD4+ T cells, CD8+ T cells, CD4+ regulatory T cells (Tregs), and NK lineage cells. FIG. 15C provides the spatial distribution of Tregs. FIG. 15D provides the spatial distribution of CD4+ T cells. FIG. 15E provides the spatial distribution of selected genes within a magnified area in human breast cancer, where CD4 (green), CD8A (blue), FOXP3 (red), NCR1 (yellow), and CTLA4 (white) are shown. Note that FOXP3-positive Tregs express T cell exhaustion markers (CTLA4 marked with red arrowheads, NK cells marked with yellow arrows).

Claims

1. 1. A method for anchoring target nucleic acids in a matrix and removing non-target cellular components, comprising: a. contacting a formalin-fixed, paraffin-embedded (FFPE) tissue sample with at least two anchoring agents, wherein a first anchoring agent forms a covalent bond with said target nucleic acid and a second anchoring agent comprises an oligonucleotide that hybridizes with said target nucleic acid; b. embedding the sample in a polymer matrix, wherein the first and second anchoring agents each form a covalent bond with the polymer matrix; and c. removing the non-target cellular components from the polymer matrix, wherein the target nucleic acid remains anchored in the polymer matrix, thereby forming a matrix-anchored target nucleic acid sample; method.

2. the first anchoring agent is an alkylating agent; The method of claim 1.

3. 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; The method of claim 2.

4. the second anchoring agent has alternating dT and locked dT moieties that hybridize with the target nucleic acid; The method of claim 1.

5. the second anchoring agent comprises a poly(dT) portion that hybridizes with the target nucleic acid; The method of claim 1.

6. the first anchoring agent and the second anchoring agent each comprise an acrydite moiety that is covalently bonded to the polymer matrix; The method of claim 1.

7. the first anchoring agent is an alkylating agent derivatized with an acrydite moiety; The method of claim 1.

8. the second anchoring agent comprises a poly(dT) moiety that hybridizes to the target nucleic acid and an acrydite moiety that covalently attaches to the polymer matrix; The method of claim 1.

9. the target nucleic acid is RNA; The method of claim 1.

10. the target nucleic acid is DNA; The method of claim 1.

11. contacting the anchored target nucleic acid sample with one or more primary oligonucleotide probes that hybridize to the target nucleic acid. The method of claim 1.

12. the primary oligonucleotide probe is a single molecule (sm) FISH probe or a multiplex error-robust fluorescence in situ hybridization (MERFISH) probe; The method of claim 11.

13. the one or more primary oligonucleotide probes comprise a first portion comprising a target sequence and a second portion comprising one or more lead sequences; The method of claim 11.

14. determining a lead sequence based on contacting the one or more primary oligonucleotide probes with a plurality of secondary nucleic acid probes comprising recognition sequences that hybridize to the lead sequence of the primary nucleic acid probe. The method of claim 13.

15. and imaging using multiplexed fluorescence in situ hybridization, comprising contacting the anchored target nucleic acid sample with one or more primary oligonucleotide probes that hybridize to the target nucleic acid, and comprising one or more successive steps of adding a plurality of secondary nucleic acid probes that include labeling moieties. The method of claim 1.

16. and imaging using multiplexed error-robust fluorescence in situ hybridization (MERFISH) probes, wherein the anchored target nucleic acid sample is contacted with one or more MERFISH probes that hybridize to the target nucleic acid. The method of claim 1.

17. further comprising imaging using multiple rounds of fluorescent in situ hybridization, in each round simultaneously reading out multiple readout sequences using one or more different secondary nucleic acid probes, each conjugated to a spectrally distinct fluorescent label; The method of claim 13.

18. 1. A method for anchoring target RNA in a matrix and removing non-target cellular components, comprising: a. contacting a formalin-fixed, paraffin-embedded (FFPE) tissue sample with at least two anchoring agents, wherein a first anchoring agent comprises an alkylating agent that forms a covalent bond with the target nucleic acid, and a second anchoring agent comprises a poly-T sequence that is complementary to and hybridizes to the target RNA, and wherein the first anchoring agent and the second anchoring agent each comprise an acrydite moiety that is covalently bound to a matrix; b. embedding the sample in a polymer matrix, wherein the first and second anchoring agents each form a covalent bond with the polymer matrix; and c. removing the non-target cellular components from the polymer matrix, wherein the target RNA remains anchored to the polymer matrix, thereby forming a matrix-anchored target RNA sample; The method of claim 1.

19. 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; 20. The method of claim 18.

20. the second anchoring agent has alternating dT and locked dT segments that hybridize to the target RNA; 20. The method of claim 18.

21. contacting the anchored target nucleic acid sample with one or more primary oligonucleotide probes that hybridize to the target nucleic acid.

20. The method of claim 18.

22. the primary oligonucleotide probe is a single molecule (sm) FISH probe or a multiplex error-robust fluorescence in situ hybridization (MERFISH) probe; 22. The method of claim 21.

23. the one or more primary oligonucleotide probes comprise a first portion comprising a target sequence and a second portion comprising one or more lead sequences; 22. The method of claim 21.

24. determining a lead sequence based on contacting the one or more primary oligonucleotide probes with a plurality of secondary nucleic acid probes comprising recognition sequences that hybridize to the lead sequence of the primary nucleic acid probe.

24. The method of claim 23.

25. and imaging using multiplexed fluorescence in situ hybridization, comprising contacting the anchored target nucleic acid sample with one or more primary oligonucleotide probes that hybridize to the target nucleic acid, and comprising one or more successive steps of adding a plurality of secondary nucleic acid probes that include labeling moieties.

20. The method of claim 18.

26. and imaging using multiplexed error-robust fluorescence in situ hybridization (MERFISH) probes, wherein the anchored target nucleic acid sample is contacted with one or more MERFISH probes that hybridize to the target nucleic acid.

20. The method of claim 18.

27. further comprising imaging using multiple rounds of fluorescent in situ hybridization, wherein in each round, multiple readout sequences are simultaneously read out using one or more different secondary nucleic acid probes, each conjugated to a spectrally distinct fluorescent label; 24. The method of claim 23.

28. 1. A method for imaging a target nucleic acid within a matrix and removing non-target cellular components, comprising: a. contacting a formalin-fixed, paraffin-embedded (FFPE) tissue sample with at least two anchoring agents, wherein a first anchoring agent forms a covalent bond with said target nucleic acid and a second anchoring agent comprises an oligonucleotide that hybridizes with said target nucleic acid; b. embedding the sample in a polymer matrix, wherein the first and second anchoring agents each form a covalent bond with the polymer matrix; c. removing the non-target cellular components from the polymer matrix, leaving the target nucleic acid anchored in the polymer matrix, thereby forming a matrix-anchored target nucleic acid sample; and d. contacting the anchored target nucleic acid sample with one or more primary oligonucleotide probes that hybridize to the target nucleic acid and a plurality of secondary nucleic acid probes that comprise fluorescent labels and recognition sequences that hybridize to sequences of the primary nucleic acid probes, and imaging the target nucleic acid; method.

29. the first anchoring agent is an alkylating agent; 29. The method of claim 28.

30. 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; 30. The method of claim 29.

31. the second anchoring agent comprises alternating dT and locked dT moieties that hybridize with the target nucleic acid; 29. The method of claim 28.

32. the second anchoring agent comprises a poly(dT) portion that hybridizes with the target nucleic acid; 29. The method of claim 28.

33. the first anchoring agent and the second anchoring agent each comprise an acrydite moiety that is covalently bonded to the polymer matrix; 29. The method of claim 28.

34. the first anchoring agent is an alkylating agent derivatized with an acrydite moiety; 29. The method of claim 28.

35. the second anchoring agent comprises a poly(dT) moiety that hybridizes to the target nucleic acid and an acrydite moiety that covalently attaches to the polymer matrix; 29. The method of claim 28.

36. the target nucleic acid is RNA; 29. The method of claim 28.

37. the target nucleic acid is DNA; 29. The method of claim 28.

38. the primary oligonucleotide probe is a single molecule (sm) FISH probe or a multiplex error-robust fluorescence in situ hybridization (MERFISH) probe; 29. The method of claim 28.

39. the one or more primary oligonucleotide probes comprise a first portion comprising a target sequence and a second portion comprising one or more lead sequences; 29. The method of claim 28.

40. determining a lead sequence based on contacting the one or more primary oligonucleotide probes with a plurality of secondary nucleic acid probes comprising recognition sequences that hybridize to the lead sequence of the primary nucleic acid probe.

40. The method of claim 39.

41. the secondary nucleic acid probe is added in one or more successive steps, and imaging is performed between each successive round of adding the secondary nucleic acid probe; 29. The method of claim 28.

42. and imaging using multiplexed error-robust fluorescence in situ hybridization (MERFISH) probes, wherein the anchored target nucleic acid sample is contacted with one or more MERFISH probes that hybridize to the target nucleic acid.

29. The method of claim 28.

43. further comprising imaging using multiple rounds of fluorescent in situ hybridization, in each round simultaneously reading out multiple readout sequences using one or more different secondary nucleic acid probes, each conjugated to a spectrally distinct fluorescent label; 41. The method of claim 40.

44. 1. A method for imaging target RNA within a matrix and removing non-target cellular components, comprising: a. contacting a formalin-fixed, paraffin-embedded (FFPE) tissue sample with at least two anchoring agents, wherein a first anchoring agent comprises an alkylating agent that forms a covalent bond with a target nucleic acid, and a second anchoring agent comprises a poly-T sequence that is complementary to and hybridizes to the target RNA, and wherein the first anchoring agent and the second anchoring agent each comprise an acrydite moiety that is covalently bound to the matrix; b. embedding the sample in a polymer matrix, wherein the first and second anchoring agents each form a covalent bond with the polymer matrix; c. removing the non-target cellular components from the polymer matrix, leaving the target RNA anchored in the polymer matrix, thereby forming a matrix-anchored target RNA sample; and d. contacting the anchored target RNA sample with one or more primary oligonucleotide probes that hybridize to the target RNA and a plurality of secondary nucleic acid probes that comprise fluorescent labels and recognition sequences that hybridize to sequences of the primary nucleic acid probes, and imaging the target nucleic acid; method.

45. 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; 45. The method of claim 44.

46. the second anchoring agent comprises alternating dT and locked dT moieties that hybridize with the target nucleic acid; 45. The method of claim 44.

47. the primary oligonucleotide probe is a single molecule (sm) FISH probe or a multiplex error-robust fluorescence in situ hybridization (MERFISH) probe; 45. The method of claim 44.

48. the one or more primary oligonucleotide probes comprise a first portion comprising a target sequence and a second portion comprising one or more lead sequences; 45. The method of claim 44.

49. determining a lead sequence based on contacting the one or more primary oligonucleotide probes with a plurality of secondary nucleic acid probes comprising recognition sequences that hybridize to the lead sequence of the primary nucleic acid probe.

49. The method of claim 48.

50. the secondary nucleic acid probe is added in one or more successive steps, and imaging is performed between each successive round of adding the secondary nucleic acid probe; 45. The method of claim 44.

51. and imaging using multiplexed error-robust fluorescence in situ hybridization (MERFISH) probes, comprising contacting the anchored target nucleic acid sample with one or more MERFISH probes that hybridize to the target nucleic acid.

45. The method of claim 44.

52. further comprising imaging using multiple rounds of fluorescent in situ hybridization, in each round simultaneously reading out multiple readout sequences using one or more different secondary nucleic acid probes, each conjugated to a spectrally distinct fluorescent label; 50. The method of claim 49.

53. the tissue sample is deparaffinized and rehydrated prior to step (a); 53. The method of any one of claims 1 to 52.

54. the polymer matrix comprises polyacrylamide; 53. The method of any one of claims 1 to 52.

55. the step (b) of embedding the sample comprises polymerizing the polymer matrix within the tissue sample, and the first and second anchoring agents each copolymerize with the polymer matrix; 53. The method of any one of claims 1 to 52.

56. The non-target cell components include proteins, lipids, DNA, RNA, or extracellular matrix.

53. The method of any one of claims 1 to 52.

57. removing the non-target cellular components comprises removing or degrading proteins from the sample; 53. The method of any one of claims 1 to 52.

58. removing the non-target cellular components comprises removing or degrading lipids from the sample; 53. The method of any one of claims 1 to 52.

59. removing the non-target cellular components includes removing or degrading non-target DNA from the sample; 53. The method of any one of claims 1 to 52.

60. removing the non-target cellular components includes removing or degrading extracellular matrix from the sample; 53. The method of any one of claims 1 to 52.

61. removing the non-target cellular components comprises exposing the sample to an enzyme capable of degrading proteins; 53. The method of any one of claims 1 to 52.

62. removing the non-target cellular components comprises exposing the sample to an enzyme capable of degrading DNA; 53. The method of any one of claims 1 to 52.

63. removing the non-target cellular components comprises exposing the sample to an enzyme capable of degrading RNA; 53. The method of any one of claims 1 to 52.

64. removing the non-target cellular components comprises exposing the sample to an enzyme capable of degrading sugars or sugar-modified biomolecules; 53. The method of any one of claims 1 to 52.

65. removing the non-target cellular components comprises exposing the sample to a detergent; 53. The method of any one of claims 1 to 52.

66. removing the non-target cellular components comprises exposing the gel to a proteinase; 53. The method of any one of claims 1 to 52.

67. The proteinase comprises proteinase K.

67. The method of claim 66.

68. removing the non-target cellular components comprises exposing the polymer matrix to guanidine HCl; 53. The method of any one of claims 1 to 52.

69. removing the non-target cellular components comprises exposing the gel to Triton X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether); 53. The method of any one of claims 1 to 52.

70. removing the non-target cellular components comprises exposing the polymer matrix to sodium dodecyl sulfate; 53. The method of any one of claims 1 to 52.

71. removing the non-target cellular components comprises exposing the polymer matrix to ethylenediaminetetraacetic acid; 53. The method of any one of claims 1 to 52.

72. further comprising amplification of the primary oligonucleotide probes, 53. The method of any one of claims 28 to 52.

73. Imaging includes using epifluorescence microscopy, total internal reflection microscopy, highly inclined thin-illumination (HILO) microscopy, light sheet microscopy, scanning confocal microscopy, scanning line confocal microscopy, spinning disk confocal microscopy, or other equivalent conventional microscopy techniques; 53. The method of any one of claims 28 to 52.

74. Imaging using multiplexed fluorescent in situ hybridization, 74. The method of claim 73.

75. the primary oligonucleotide probes are present in pools of probes, each pool hybridizing to a distinct nucleic acid target, and each pool of probes encoding, via a read sequence, an N-bit binary code assigned to each distinct nucleic acid target; 53. The method of any one of claims 28 to 52.

76. the N-bit binary code has a Hamming weight of at least 2; 76. The method of claim 75.

77. the integrity of the nucleic acid is determined prior to step a), 53. The method of any one of claims 1 to 52.

Images