Clinical- and industrial-scale intact-tissue sequencing

Abstract

To provide methods of enhancing the robustness, rapidity, automation, and high-throughput nature of in situ sequencing techniques.SOLUTION: Provided herein are devices, methods and systems for in situ gene sequencing of a target nucleic acid in a cell in an intact tissue. Methods of screening a candidate agent to determine whether the candidate agent modulates gene expression of a nucleic acid in a cell in an intact tissue are also provided herein.SELECTED DRAWING: Figure 1-1

Description

[Background technology]

[0001] Biological samples contain complex and heterogeneous genetic information spanning the length scale of individual cells to entire tissues. Spatial patterns of nucleic acids within cells can reveal characteristics and abnormalities of cellular function, the cumulative distribution of RNA expression can define cell type or function, and systematic variations in the location of cell types within tissues can define tissue function. The combination of anatomical connectivity information encoded in nucleic acids and the distribution of cell types throughout tissues can span many sections of the tissue.

[0002] Therefore, techniques for in situ nucleic acid sequencing must be able to bridge resolutions as small as individual molecules and as large as the entire brain. Efficiently collecting and recording this information across orders of magnitude of length requires new inventions to enhance the robustness, rapidity, automation, and high-throughput nature of in situ sequencing techniques. Summary of the Invention

[0003] Devices, methods, and systems are provided for in situ gene sequencing of target nucleic acids in cells in intact tissue. Also provided herein are methods for screening candidate agents to determine whether the candidate agents modulate gene expression of nucleic acids in cells in intact tissue.

[0004] In one aspect, a method is provided for in situ gene sequencing of a target nucleic acid in cells in an intact tissue, the method comprising: (a) contacting a fixed and permeabilized intact tissue sample with at least a pair of oligonucleotides under conditions that allow for specific hybridization, the pair of oligonucleotides comprising a first oligonucleotide and a second oligonucleotide, each of the first oligonucleotide and the second oligonucleotide comprising a first region of complementarity, a second region of complementarity, and a third region of complementarity; The second oligonucleotide further comprises a barcode sequence, wherein a first complementary region of the first oligonucleotide is complementary to a first portion of the target nucleic acid, a second complementary region of the first oligonucleotide is complementary to a first complementary region of the second oligonucleotide, a third complementary region of the first oligonucleotide is complementary to a third complementary region of the second oligonucleotide, and a second complementary region of the second oligonucleotide is complementary to a second portion of the target nucleic acid. (b) contacting a first oligonucleotide with a second complementary region adjacent to the second complementary region of the second oligonucleotide; (b) adding a ligase to ligate the 5' and 3' ends of the second oligonucleotide to generate a closed nucleic acid circle; (c) pre-incubating the tissue sample with DNA polymerase for a time sufficient to allow uniform diffusion of the DNA polymerase throughout the tissue sample before performing rolling circle amplification; (d) performing rolling circle amplification by contacting the tissue sample with deoxyribonucleotide triphosphates to generate one or more amplicons, wherein the ligated second oligonucleotide serves as a template and the first oligonucleotide serves as a primer for the DNA polymerase; (e) embedding the tissue sample in a hydrogel before or after any one of steps (a)-(d); (f) crosslinking the one or more amplicons to the hydrogel; (g) purifying the hydrogel to enhance the transparency of the hydrogel; and (h) isolating the one or more hydrogel-embedded amplicons having the barcode sequence.contacting a lead primer and a fluorescently labeled probe, wherein the probe comprises a first thiol covalently attached to a fluorophore comprising a second thiol group by a disulfide bond between the first thiol group and the second thiol group; (i) contacting a hydrogel with an anti-fade buffer containing an oligonucleotide containing a methyl group; (i) ligating a lead primer and a fluorescently labeled probe, wherein ligation occurs only if both the lead primer and the fluorescently labeled probe are complementary to adjacent sequences of the same amplicon; (j) contacting the hydrogel with an anti-fade buffer containing an antioxidant; (k) imaging one or more hydrogel-embedded amplicons to determine the location of target nucleic acids in cells in an intact tissue undergoing in situ gene sequencing, wherein the imaging is performed in the presence of the anti-fade buffer; (l) contacting the hydrogel with a reducing agent, resulting in reduction of disulfide bonds and cleavage of the fluorophore from the probe; (m) removing the fluorophore from the hydrogel; and (n) repeatedly performing steps (h)-(m).

[0005] In certain embodiments, the method further comprises sectioning the intact tissue and placing the section of intact tissue flat on a surface. For example, a metal ring can be used to press down on the section and transfer the section flat onto the surface. In some embodiments, cryosectioning is performed on the tissue.

[0006] In another aspect, a fluidic system for automation of the methods described herein is provided that allows for continuous operation, hi some embodiments, the system comprises a fluidic device and a processor configured to perform the methods described herein.

[0007] In certain embodiments, a fluidic system is provided that includes: a) a device including a non-pressurized sample chamber; b) a lid that covers the top of the sample chamber; c) an interface tube, the first end of which is held by the lid and the second end of which is positioned above the tissue sample in the sample chamber, sufficiently close to the bottom edge of the sample chamber so that liquid in the sample chamber remains in contact with the bottom of the interface tube as long as the liquid remains in the sample chamber; d) a fluid line coupled to the first end of the interface tube such that fluid flows through the fluid line into the interface tube and out the second end of the interface tube onto the tissue sample in the sample chamber; e) a pump; f) an imaging system; and g) a processor unit configured to perform the methods described herein. In some embodiments, the sample chamber is a well of a multiwell plate or slide chamber. In some embodiments, the interface tube is positioned at a polar angle relative to the tissue sample plane. In some embodiments, the fluidic system further includes a cryostat that maintains the sample chamber at a cryogenic temperature. In some embodiments, the fluidic system further comprises a multi-way selector valve interfaced with the pump. In some embodiments, the fluidic system further comprises a reagent tray comprising one or more containers or wells containing reagents for performing the methods described herein, the reagents being fluidly connected to the multi-way selector valve. In some embodiments, the fluidic system further comprises a buffer tray comprising one or more containers or wells containing buffers for performing the methods described herein, the buffers being fluidly connected to the multi-way selector valve. In some embodiments, the reagent tray and / or buffer tray are disposable. In some embodiments, the fluidic system further comprises an adjustable stage supporting the reagent tray, a cooler, and a position sensor enabling movement of the adjustable stage to enable selection of a reagent from the reagent tray.In some embodiments, the fluidic system further comprises a waste container fluidly connected to the multi-way selector valve. In some embodiments, the imaging system comprises a microscope that performs confocal microscopy, spinning disk confocal microscopy, light sheet microscopy, lattice light sheet microscopy, or light field microscopy. In some embodiments, the fluidic system further comprises a container surrounding the fluidic system, the container blocking ambient light. In some embodiments, In embodiments, the enclosure surrounding the fluid system comprises one or more doors or drawers. In some embodiments, the enclosure surrounding the fluid system is an acoustically attenuating enclosure. [Brief explanation of the drawings]

[0008] [Figure 1-1] Figure 1 shows intact tissue sequencing integrating anatomical and genomic data. [Figure 1-2] Figure 1 shows intact tissue sequencing integrating anatomical and genomic data. [Figure 2] 1 illustrates the challenges in sequencing intact tissues. [Figure 3] The advantages of the CISITS method are shown. [Figure 4] Anti-bleaching with propyl gallate or other antioxidants is shown to reduce photobleaching. [Figure 5A] Showing round-to-round robustness using thiol-mediated readout fluoroprobes for sequential sequencing. [Figure 5B] Showing round-to-round robustness using thiol-mediated readout fluoroprobes for sequential sequencing. [Figure 5C] Showing round-to-round robustness using thiol-mediated readout fluoroprobes for sequential sequencing. [Figure 6] 1 shows combinatorial sequencing in thick tissue. [Figure 7-1] A diagram of the CISITS sequencing device is shown. [Figure 7-2] A diagram of the CISITS sequencing device is shown. [Figure 8] CISITS software interface. DETAILED DESCRIPTION OF THE INVENTION

[0009] Provided herein are devices, methods, and systems for in situ gene sequencing of target nucleic acids in cells in intact tissue. Also provided herein are methods for screening candidate agents to determine whether the candidate agents modulate gene expression of nucleic acids in cells in intact tissue.

[0010] Before the present devices, methods, and systems are described, it is to be understood that this invention is not limited to the particular methods or compositions described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, since the scope of the present invention will be limited only by the appended claims.

[0011] Where a range of values ​​is provided, unless the context dictates otherwise, it is understood that each intervening value between the upper and lower limit of that range is also specifically disclosed to the tenth of the unit of the lower limit. Each smaller range between any stated value or intervening value within a stated range and any other stated value or intervening value within that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded, and each range in which either, neither, or both limits are included in the smaller range is also encompassed within the invention, subject to any specifically excluded limits in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the invention.

[0012] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. In case of conflict, it is understood that the present disclosure supersedes any disclosure of the incorporated publications.

[0013] As will be apparent to those skilled in the art upon reading this disclosure, each of the separate embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other various embodiments without departing from the scope or spirit of the invention. Any recited method may be carried out in the order of events recited or in any other order which is logically possible.

[0014] It should 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. Thus, for example, a reference to "a cell" includes a plurality of such cells, and a reference to "the peptide" includes a reference to one or more peptides and equivalents thereof known to those skilled in the art, such as oligopeptides or polypeptides.

[0015] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0016] definition The term "about" is meant to encompass a deviation of plus or minus 5 percent, particularly with respect to a given quantity.

[0017] The terms "peptide," "oligopeptide," "polypeptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of corresponding naturally occurring amino acids, as well as naturally occurring and non-naturally occurring amino acid polymers. Both full-length proteins and fragments thereof are encompassed by the definition. The term also includes post-expression modifications of polypeptides, such as phosphorylation, glycosylation, acetylation, hydroxylation, oxidation, etc., as well as chemically or biochemically modified or derivatized amino acids and polypeptides having modified peptide backbones. The term also includes fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences with or without an N-terminal methionine residue, immunologically tagged proteins, and the like. The term includes polypeptides containing one or more of a fatty acid moiety, a lipid moiety, a sugar moiety, and a carbohydrate moiety.

[0018] As used herein, the term "target nucleic acid" refers to any polynucleotide nucleic acid molecule (e.g., DNA molecule, RNA molecule, modified nucleic acid, etc.) present in a single cell. In some embodiments, the target nucleic acid is a coding RNA (e.g., mRNA). In some embodiments, the target nucleic acid is a non-coding RNA (e.g., tRNA, rRNA, microRNA (miRNA), mature miRNA, immature miRNA, etc.). In some embodiments, the target nucleic acid is a splice variant of an RNA molecule (e.g., mRNA, pre-mRNA, etc.) in the context of a cell. Thus, suitable target nucleic acids include unspliced ​​RNA (e.g., pre-mRNA, mRNA), partially spliced ​​RNA, and the like. The target nucleic acid of interest may be variably expressed in a cell population, i.e., have different abundances, and the method of the present invention allows for profiling and comparison of the expression levels of nucleic acids, including but not limited to RNA transcripts, in individual cells.The target nucleic acid may also be a DNA molecule, such as a modified genome, a virus, a plasmid, etc.For example, the method can be used to detect copy number variants in cancer cell populations, where the target nucleic acid is present in different abundances in the genomes of cells in the population, or in virus-infected cells to determine viral load and kinetics.

[0019] The terms "oligonucleotide," "polynucleotide," and "nucleic acid molecule," used interchangeably herein, refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the terms include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of a polynucleotide may contain sugar and phosphate groups (typically found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of a polynucleotide may comprise a polymer of synthetic subunits, such as phosphoramidites and / or phosphorothioates, and thus may be oligodeoxynucleoside phosphoramidate or mixed phosphoramidate-phosphodiester oligomers. See Peyrottes et al. (1996) Nucl. Acids Res. 24:1841-1848, Chaturvedi et al. (1996) Nucl. Acids Res. 24:2318-2323. A polynucleotide may contain one or more L-nucleosides. A polynucleotide may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracil, other sugars, and linking groups such as fluororibose and thioate, and nucleotide branches. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be modified to contain N3'-P5' (NP) phosphoramidates, morpholinophosphorcyamidates (MF), locked nucleic acids (LNA), 2'-O-methoxyethyl (MOE), or 2'-fluoroarabinonucleic acid (FANA), which can enhance the resistance of polynucleotides to nuclease degradation (see, e.g., Faria et al. (2001) Nature Biotechnol. 19:40-44; Toulme (2001) Nature Biotechnol. 19:17-18).Polynucleotides may be further modified after polymerization, for example, by conjugation with a labeling component. Other types of modifications included in this definition include capping, substitution of one or more analogs of naturally occurring nucleotides, and the introduction of a means for binding polynucleotides to proteins, metal ions, labeling components, other polynucleotides, or solid supports. As described in more detail herein, immune modulatory nucleic acid molecules can be provided in various formulations, for example, in association with liposomes, microencapsulation, etc. Polynucleotides used for amplification are generally single-stranded for maximum amplification efficiency, but may alternatively be double-stranded. If double-stranded, the polynucleotide is first treated to separate its strands, which can then be used to prepare extension products. This denaturation step is typically affected by heat, but may alternatively be carried out using alkali, followed by neutralization.

[0020] "Isolated," when referring to a protein, polypeptide, or peptide, means that the indicated molecule is separated and free from the whole organism in which it is naturally found, or exists in the substantial absence of other biological macromolecules of the same type. With respect to polynucleotides, the term "isolated" refers to a nucleic acid molecule that lacks all or part of sequences that are normally associated with it in nature, or that is naturally occurring but does not contain heterologous sequences associated with it. It is a sequence that contains a chromosome, or a molecule that has dissociated from a chromosome.

[0021] The terms "subject," "individual," or "patient" are used interchangeably herein and refer to vertebrates, preferably mammals. "Vertebrate" refers to any member of the subphylum Chordata, including but not limited to humans and other primates, including non-human primates such as chimpanzees and other apes and monkeys; livestock animals such as cows, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals, including rodents such as mice, rats, and guinea pigs; domestic birds, including chickens, turkeys, and other poultry birds, ducks, geese, and wild birds and game birds. The term does not denote a specific age. Thus, it is intended to apply to both adult and newborn individuals.

[0022] method The methods disclosed herein involve the use of a modified spatially resolved transcription amplicon read mapping (STARmap) technique, termed "Clinical and Industrial-Scale Intact Tissue Sequencing" (CISITS), which improves both sequencing chemistry and automated sequencing execution. For a description of the original STARmap technique, see, for example, International Patent Application Publication No. 2019 / 199579A1 and Wang et al. (2018) Science 361(6400):eaat5691, which are incorporated herein by reference in their entireties. Similar to STARmap, CISITS utilizes image-based in situ nucleic acid (DNA and / or RNA) sequencing technology using a sequencing-by-ligation process, specific signal amplification, hydrogel histochemistry to convert biological tissue into a transparent sequencing chip, and an associated data analysis pipeline for spatially resolving highly multiplexed gene detection at the subcellular and cellular levels. CISITS adds improvements that increase the signal-to-noise ratio (SNR) of labeled molecules, eliminate carryover signal across rounds, and enable combinatorial sequencing in thick tissues. Also disclosed herein is a fluidic system for automation of the CISITS method that allows parallelization across samples. The fluidic system includes a sample chamber that is no longer pressurized, and also includes integration of CISITS with microscope hardware. Additionally, CISITS software is provided for controlling the automated fluidic system.

[0023] As summarized above, the method disclosed herein includes a method for in situ gene sequencing of target nucleic acids in cells in intact tissue. The method typically includes: (a) contacting a fixed and permeabilized intact tissue sample with at least a pair of oligonucleotides under conditions that allow specific hybridization, the pair of oligonucleotides comprising a first oligonucleotide and a second oligonucleotide, each of the first oligonucleotide and the second oligonucleotide comprising a first complementary region, a second complementary region, and a third complementary region, the second oligonucleotide further comprising a barcode sequence, the first complementary region of the first oligonucleotide being complementary to a first portion of the target nucleic acid, the second complementary region of the first oligonucleotide being complementary to the first complementary region of the second oligonucleotide, and the third complementary region of the first oligonucleotide being complementary to the second complementary region of the second oligonucleotide. (b) contacting a first oligonucleotide with a second oligonucleotide, the second oligonucleotide being complementary to a third complementary region of the first oligonucleotide, the second complementary region of the second oligonucleotide being complementary to a second portion of the target nucleic acid, the first complementary region of the first oligonucleotide being adjacent to the second complementary region of the second oligonucleotide; (b) adding a ligase to ligate the 5' and 3' ends of the second oligonucleotide to generate a closed nucleic acid circle; (c) pre-incubating the tissue sample with DNA polymerase for a sufficient time to allow uniform diffusion of the DNA polymerase throughout the tissue sample prior to performing rolling circle amplification; and (d) performing rolling circle amplification by contacting the tissue sample with deoxyribonucleotide triphosphates to generate one or more amplicons. (e) embedding the tissue sample in a hydrogel before or after any one of steps (a)-(d); (f) crosslinking one or more amplicons to the hydrogel; (g) purifying the hydrogel to enhance transparency of the hydrogel; (h) contacting the one or more hydrogel-embedded amplicons having the barcode sequence with a lead primer and a fluorescently labeled probe, wherein the probe comprises an oligonucleotide comprising a first thiol group covalently bonded to a fluorophore comprising a second thiol group by a disulfide bond between the first thiol group and the second thiol group; (i) (j) ligating a lead primer and a fluorescently labeled probe, wherein ligation occurs only if both the lead primer and the fluorescently labeled probe are complementary to adjacent sequences of the same amplicon; (j) contacting the hydrogel with an anti-fade buffer comprising an antioxidant; (k) imaging one or more hydrogel-embedded amplicons to determine the location of target nucleic acids in cells in an intact tissue undergoing in situ gene sequencing, wherein the imaging is performed in the presence of the anti-fade buffer; (l) contacting the hydrogel with a reducing agent, resulting in reduction of disulfide bonds and cleavage of the fluorophore from the probe; (m) removing the fluorophore from the hydrogel; and (n) repeatedly performing steps (h)-(m).

[0024] The methods disclosed herein also provide a method of screening a candidate agent to determine whether the candidate agent modulates gene expression of a nucleic acid in cells in an intact tissue, the method comprising: (a) contacting a fixed and permeabilized intact tissue sample with at least a pair of oligonucleotide primers under conditions that allow specific hybridization, the pair of primers comprising a first oligonucleotide and a second oligonucleotide, each of the first oligonucleotide and the second oligonucleotide having a first complementary sequence; a first complementary region, a second complementary region, and a third complementary region, wherein the second oligonucleotide further comprises a barcode sequence, wherein the first complementary region of the first oligonucleotide is complementary to a first portion of the target nucleic acid, the second complementary region of the first oligonucleotide is complementary to the first complementary region of the second oligonucleotide, the third complementary region of the first oligonucleotide is complementary to the third complementary region of the second oligonucleotide, and the second complementary region of the second oligonucleotide is complementary to a second portion of the target nucleic acid, and the first oligonucleotide (b) contacting a first complementary region of the nucleotides adjacent to a second complementary region of a second oligonucleotide; (b) adding a ligase to ligate the second oligonucleotide to generate a closed nucleic acid circle; (c) performing rolling circle amplification in the presence of the nucleic acid molecule, comprising forming one or more amplicons using the second oligonucleotide as a template and the first oligonucleotide as a primer for a polymerase; (d) embedding the one or more amplicons in the presence of a hydrogel subunit to form one or more hydrogel-embedded amplicons; (e) contacting the one or more hydrogel-embedded amplicons having the barcode sequence with a pair of primers under conditions that allow ligation, wherein the pair of primers comprises a third oligonucleotide and a fourth oligonucleotide, and ligation occurs only if both the third oligonucleotide and the fourth oligonucleotide ligate to the same amplicon; and (f) repeating step (e).(g) imaging the one or more hydrogel-embedded amplicons to determine in situ gene sequencing of the target nucleic acid in cells in the intact tissue; and (h) detecting a gene expression level of the target nucleic acid, wherein a change in the expression level of the target nucleic acid in the presence of the at least one candidate agent relative to the expression level of the target nucleic acid in the absence of the at least one candidate agent indicates that the at least one candidate agent modulates gene expression of the nucleic acid in cells in the intact tissue. and detecting the

[0025] In certain embodiments, the methods disclosed herein offer faster processing times, higher multiplexing (up to 1000 genes), higher efficiency, higher sensitivity, lower error rates, and more spatially resolved cell types compared to existing gene expression analysis tools. In such embodiments, the improved hydrogel histochemistry transforms biological tissue into hydrogel-imprinted nucleic acids that are compatible with in situ sequencing, which is an improved sequencing by ligation process (SEDAL) for in situ sequencing with reduced error. In some other embodiments, the methods disclosed herein include spatial sequencing (e.g., reagents, chips, or services) for biomedical research and clinical diagnostics (e.g., cancer, bacterial infection, viral infection, etc.) with single-cell and / or single-molecule sensitivity.

[0026] Specific Amplification of Nucleic Acids via Intramolecular Ligation (SNAIL) In some embodiments, one component of CISITS comprises an efficient approach for generating cDNA libraries from cellular RNA in situ for specific amplification of nucleic acids via intramolecular ligation, which may be referred to as SNAIL. In certain embodiments, the subject method comprises contacting fixed and permeabilized intact tissue with at least a pair of oligonucleotide primers under conditions that allow specific hybridization, the pair of primers comprising a first oligonucleotide and a second oligonucleotide.

[0027] More generally, the nucleic acid present in the target cell in tissue serves as a scaffold for the assembly of a complex comprising a pair of primers, herein referred to as first oligonucleotide and second oligonucleotide.In some embodiments, contacting the intact tissue that is fixed and permeabilized comprises hybridizing a pair of primers to the same target nucleic acid.In some embodiments, the target nucleic acid is RNA.In such embodiments, the target nucleic acid can be mRNA.In other embodiments, the target nucleic acid is DNA.

[0028] As used herein, the terms "hybridize" and "hybridization" refer to the formation of a complex between nucleotide sequences that are sufficiently complementary to form a complex through Watson-Crick base pairing. When a primer "hybridizes" with a target (template), such a complex (or hybrid) is sufficiently stable to perform the priming function required, for example, by DNA polymerase, to initiate DNA synthesis. It is understood that hybridizing sequences do not need to have perfect complementarity to provide a stable hybrid. In many cases, stable hybrids are formed with less than about 10% mismatch of bases, ignoring loops of four or more nucleotides. Therefore, as used herein, the term "complementary" refers to an oligonucleotide that forms a stable duplex with its "complement" under assay conditions, generally with about 90% or more homology.

[0029] SNAIL oligonucleotide primers In the method of the present invention, the SNAIL oligonucleotide primer comprises at least a first oligonucleotide and a second oligonucleotide, each of the first oligonucleotide and the second oligonucleotide comprising a first complementary region, a second complementary region, and a third complementary region, the second oligonucleotide further comprising a barcode sequence, the first complementary region of the first oligonucleotide being complementary to a first portion of the target nucleic acid, the second complementary region of the first oligonucleotide being complementary to the first complementary region of the second oligonucleotide, and the third complementary region of the first oligonucleotide being complementary to the second complementary region. In an alternative embodiment, the second oligonucleotide is a closed circular molecule, and the ligation step is omitted.

[0030] The present disclosure provides a method for contacting a fixed and permeabilized tissue with a plurality of oligonucleotide primers having specificity for different target nucleic acids. In some embodiments, the method includes a plurality of first oligonucleotides, including but not limited to, 5 or more, for example, 8 or more, 10 or more, 12 or more, 15 or more, 18 or more, 20 or more, 25 or more, 30 or more, 35 or more, that hybridize to target nucleotide sequences. In some embodiments, the method of the present disclosure includes a plurality of first oligonucleotides, including but not limited to, 15 or more, for example, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, and up to 80 different target nucleotide sequences that hybridize to 15 or more, for example, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, and up to 80 different first oligonucleotides. In some embodiments, the method comprises a plurality of second oligonucleotides, including but not limited to, 5 or more second oligonucleotides, for example, 8 or more, 10 or more, 12 or more, 15 or more, 18 or more, 20 or more, 25 or more, 30 or more, 35 or more. In some embodiments, the method of the present disclosure comprises a plurality of second oligonucleotides, including but not limited to, 15 or more second oligonucleotides, for example, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, and up to 80 different first oligonucleotides, hybridizing to 15 or more different target nucleotide sequences, for example, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, and up to 80 different first oligonucleotides. Multiple oligonucleotide pairs can be used in the reaction, and one or more pairs specifically bind to each target nucleic acid. For example, to improve sensitivity and reduce variability, two primer pairs can be used for one target nucleic acid.It is also of interest to detect multiple different target nucleic acids in a cell, for example, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 12, up to 15, up to 18, up to 20, up to 25, up to 30, up to 40 or more different target nucleic acids. Primers are typically denatured prior to use, typically by heating to a temperature of at least about 50°C, at least about 60°C, at least about 70°C, at least about 80°C, and up to about 99°C, up to about 95°C, up to about 90°C.

[0031] In some embodiments, the primers are denatured by heating before contacting the sample. In certain aspects, the melting temperature (T m ) is chosen to minimize ligation in solution. The "melting temperature" or "T" of a nucleic acid m " is defined as the temperature at which half of the helical structure of a nucleic acid is lost due to heating or other dissociation of hydrogen bonds between base pairs, for example, by acid or alkali treatment. m depends on its length and base composition. Nucleic acid molecules rich in GC base pairs have a higher T than those rich in AT base pairs. m The complementary strands of the separated nucleic acids are m When the temperature drops below T, they spontaneously reassociate or anneal to form double-stranded nucleic acids. The maximum rate of nucleic acid hybridization is T m This occurs approximately 25°C below T m can be estimated using the following relationship: T m =69.3+0.41(GC)% (Marmur et al. (1962) J. Mol. Biol. 5:109-118).

[0032] In certain embodiments, the plurality of second oligonucleotides comprises padlock probes. In some embodiments, the probe comprises a detectable label that can be measured and quantified. The terms "label" and "detectable label" refer to a detectable molecule, including, but not limited to, a radioisotope, a fluorophore, a chemiluminescent material, an enzyme, an enzyme substrate, an enzyme cofactor, an enzyme inhibitor, a chromophore, a dye, a metal ion, a metal sol, a ligand (e.g., biotin or a hapten), and the like. The term "fluorescent agent" refers to a substance or portion thereof that can exhibit fluorescence within the detectable range. Specific examples of labels that can be used with the present invention include, but are not limited to, phycoerythrin, Alexa dyes, fluorescein, YPet, CyPet, Cascade Blue, allophycocyanin, Cy3, Cy5, Cy7, rhodamine, dansyl, umbelliferone, Texas Red, luminol, acridinium esters, biotin, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), firefly luciferase, renilla luciferase, NADPH, beta-galactosidase, horseradish peroxidase, glucose oxidase, alkaline phosphatase, chloramphenicol acetyltransferase, and urase.

[0033] In some embodiments, one or more first and second oligonucleotides bind to different regions of a target nucleic acid or target site. Each target site in a pair is different, and the target sites are adjacent sites on the target nucleic acid, e.g., typically no more than 15 nucleotides from the other site, e.g., no more than 10, 8, 6, 4, or 2 nucleotides away, and may be contiguous. The target sites are typically located on the same strand of the target nucleic acid in the same orientation. The target sites are also selected to provide unique binding sites for other nucleic acids present in the cell. Each target site is generally about 19 to about 25 nucleotides in length, e.g., about 19 to 23 nucleotides, about 19 to 21 nucleotides, or about 19 to 20 nucleotides. The first and second oligonucleotide pairs are selected so that each oligonucleotide in the pair has a similar melting temperature for binding to its cognate target site, e.g., T m can be from about 50° C., from about 52° C., from about 55° C., from about 58° C., from about 62° C., from about 65° C., from about 70° C., or from about 72° C. The GC content of the target site can generally be selected to be about 20% or less, about 30% or less, about 40% or less, about 50% or less, about 60% or less, about 70% or less.

[0034] In some embodiments, the first oligonucleotide comprises a first, second, and third complementary region. The target site of the first oligonucleotide may refer to the first complementary region. As summarized above, the first complementary region of the first oligonucleotide may have a length of 19 to 25 nucleotides. In certain aspects, the second complementary region of the first oligonucleotide has a length of 3 to 10 nucleotides, including, for example, 4 to 8 nucleotides or 4 to 7 nucleotides. In some aspects, the second complementary region of the first oligonucleotide has a length of 6 nucleotides. In some embodiments, the third complementary region of the first oligonucleotide also has a length of 6 nucleotides. In such embodiments, the third complementary region of the first oligonucleotide has a length of 3 to 10 nucleotides, including, for example, 4 to 8 nucleotides or 4 to 7 nucleotides.

[0035] In some embodiments, the second oligonucleotide comprises a first, second, and third complementary region. The target site of the second oligonucleotide may refer to the second complementary region. As summarized above, the second complementary region of the second oligonucleotide may have a length of 19 to 25 nucleotides. In certain aspects, the first complementary region of the first oligonucleotide has a length of 3 to 10 nucleotides, including, for example, 4 to 8 nucleotides or 4 to 7 nucleotides. In some aspects, the first complementary region of the first oligonucleotide has a length of 6 nucleotides. In some aspects, the first complementary region of the second oligonucleotide comprises the 5' end of the second oligonucleotide.

[0036] In some embodiments, the third complementary region of the second oligonucleotide similarly has a length of 6 nucleotides. In such embodiments, the third complementary region of the second oligonucleotide has a length of 3 to 10 nucleotides, including, for example, 4 to 8 nucleotides or 4 to 7 nucleotides. In further embodiments, the third complementary region of the second oligonucleotide comprises the 3' end of the second oligonucleotide. In some embodiments, the first complementary region of the second oligonucleotide is adjacent to the third complementary region of the second oligonucleotide.

[0037] In some embodiments, the second oligonucleotide comprises a barcode sequence, and the barcode sequence of the second oligonucleotide provides barcode information for identifying the target nucleic acid. The term "barcode" refers to a nucleic acid sequence used to identify a single cell or a subpopulation of cells. The barcode sequence can be linked to the target nucleic acid of interest during amplification and used to trace the amplicon to the cell from which the target nucleic acid originated. The barcode sequence can be added to the target nucleic acid of interest during amplification by performing amplification with oligonucleotides containing a region including the barcode sequence and a region complementary to the target nucleic acid such that the barcode sequence is ultimately incorporated into the amplified target nucleic acid product (i.e., amplicon).

[0038] organization As described herein, the disclosed methods include in situ sequencing techniques by contacting fixed and permeabilized intact tissue with at least one pair of oligonucleotide primers under conditions that allow specific hybridization. Tissue specimens suitable for use with the methods described herein generally include any type of tissue specimen collected from a living or cadaveric subject, such as biopsy and autopsy specimens, including, but not limited to, epithelial, muscle, connective, and neural tissue. Tissue specimens may be collected and processed using the methods described herein and subjected to microscopic analysis immediately after processing, or may be preserved and subjected to microscopic analysis at a future time, for example, after long-term storage. In some embodiments, the methods described herein may be used to preserve tissue specimens in a stable, accessible, and fully intact form for future analysis. In some embodiments, the methods described herein may be used to analyze previously preserved or archived tissue specimens. In some embodiments, the intact tissue includes brain tissue, such as visual cortex slices. In some embodiments, the intact tissue is a thin slice having a thickness of 5-20 μm, for example, but not limited to, 5-18 μm, 5-15 μm, or 5-10 μm, while in other embodiments, the intact tissue is a thick slice having a thickness of 50-200 μm, for example, but not limited to, 50-150 μm, 50-100 μm, or 50-80 μm.

[0039] Embodiments of the present invention include fixating intact tissue. As used herein, the term "fixing" or "fixation" refers to the process of preserving biological materials (e.g., tissues, cells, organelles, molecules, etc.) from decay and / or degradation. Fixation can be achieved using any convenient protocol. Fixation can include contacting the sample with a fixation reagent (i.e., a reagent containing at least one fixation agent). The sample can be contacted with the fixation reagent for a wide range of times, which can depend on the temperature, the nature of the sample, and the fixation agent. For example, the sample can be contacted with the fixation reagent for 24 hours or less, 18 hours or less, 12 hours or less, 8 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, 60 minutes or less, 45 minutes or less, 30 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, or 2 minutes or less.

[0040] The sample is allowed to stand for 5 minutes to 24 hours, for example, 10 minutes to 20 hours, 10 minutes to 18 hours, 10 minutes to 12 hours, 10 minutes to 8 hours, 10 minutes to 6 hours, 10 minutes to 4 hours, 10 minutes to 2 hours, 15 minutes to The immobilization reagent may be contacted for a period ranging from 20 hours, 15 minutes to 18 hours, 15 minutes to 12 hours, 15 minutes to 8 hours, 15 minutes to 6 hours, 15 minutes to 4 hours, 15 minutes to 2 hours, 15 minutes to 1.5 hours, 15 minutes to 1 hour, 10 minutes to 30 minutes, 15 minutes to 30 minutes, 30 minutes to 2 hours, 45 minutes to 1.5 hours, or 55 minutes to 70 minutes.

[0041] The sample may be contacted with the immobilization reagent at a variety of temperatures, depending on the protocol and reagents used. For example, in some instances, the sample may be contacted with the immobilization reagent at temperatures ranging from -22°C to 55°C, with particular ranges of interest including, but not limited to, 50-54°C, 40-44°C, 35-39°C, 28-32°C, 20-26°C, 0-6°C, and -18 to -22°C. In some cases, the sample may be contacted with the immobilization reagent at temperatures of -20°C, 4°C, room temperature (22-25°C), 30°C, 37°C, 42°C, or 52°C.

[0042] Any convenient fixation reagent can be used. Common fixation reagents include cross-linking fixation agents, precipitation fixation agents, oxidative fixation agents, mercury, and the like. Cross-linking fixation agents chemically link two or more molecules by a covalent bond, and a wide variety of cross-linking reagents can be used. Examples of suitable cross-linking fixation agents include, but are not limited to, aldehydes (e.g., formaldehyde, also commonly referred to as "paraformaldehyde" and "formalin," glutaraldehyde, and the like), imidoesters, NHS (N-hydroxysuccinimide) esters, and the like. Examples of suitable precipitation fixation agents include, but are not limited to, alcohols (e.g., methanol, ethanol, and the like), acetone, acetic acid, and the like. In some embodiments, the fixation agent is formaldehyde (i.e., paraformaldehyde or formalin). Suitable final concentrations of formaldehyde in the fixation reagent are 0.1-10%, 1-8%, 1-4%, 1-2%, 3-5%, or 3.5-4.5%, including approximately 1.6%, for 10 minutes. In some embodiments, samples are fixed at a final concentration of 4% formaldehyde (diluted from a more concentrated stock solution, e.g., 38%, 37%, 36%, 20%, 18%, 16%, 14%, 10%, 8%, 6%, etc.). In some embodiments, samples are fixed at a final concentration of 10% formaldehyde. In some embodiments, samples are fixed at a final concentration of 1% formaldehyde. In some embodiments, the fixative is glutaraldehyde. A suitable concentration of glutaraldehyde in the fixation reagent is 0.1-1%. The fixation reagent may contain two or more fixatives in any combination. For example, in some embodiments, the sample is contacted with a fixation reagent that contains both formaldehyde and glutaraldehyde.

[0043] As used herein, the terms "permeabilization" or "permeabilizing" refer to the process of rendering cells (e.g., cell membranes) of a sample permeable to experimental reagents, such as nucleic acid probes, antibodies, and chemical substrates. Any convenient method and / or reagent for permeabilization can be used. Suitable permeabilization reagents include detergents (e.g., saponin, Triton X-100, Tween-20, etc.), organic fixatives (e.g., acetone, methanol, ethanol, etc.), enzymes, etc. Detergents can be used in a wide range of concentrations. For example, 0.001% to 1% detergent, 0.05% to 0.5% detergent, or 0.1% to 0.3% detergent can be used for permeabilization (e.g., 0.1% saponin, 0.2% Tween-20, 0.1% to 0.3% Triton X-100, etc.). In some embodiments, permeabilization is performed using methanol on ice for at least 10 minutes.

[0044] In some embodiments, the same solution may be used as both the fixation reagent and the permeabilization reagent. For example, in some embodiments, the fixation reagent contains 0.1% to 10% formaldehyde and 0.001% to 1% saponin. In some embodiments, the fixation reagent contains 1% formaldehyde and 0.3% saponin.

[0045] The sample may be contacted with the permeabilization reagent for a wide range of times, which may depend on the temperature, the nature of the sample, and the permeabilization reagent. For example, the sample may be contacted with the permeabilization reagent for 24 hours or more, 24 hours or less, 18 hours or less, 12 hours or less, 8 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, 60 minutes or less, 45 minutes or less, 30 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, or 2 minutes or less. The sample may be contacted with the permeabilization reagent at various temperatures, depending on the protocol and reagents used. For example, in some instances, the sample may be contacted with the permeabilization reagent at temperatures ranging from -82°C to 55°C, with particular ranges of interest including, but not limited to, 50-54°C, 40-44°C, 35-39°C, 28-32°C, 20-26°C, 0-6°C, -18°C to -22°C, and -78°C to -82°C. In some examples, the sample may be contacted with the permeabilization reagent at a temperature of -80°C, -20°C, 4°C, room temperature (22-25°C), 30°C, 37°C, 42°C, or 52°C.

[0046] In some embodiments, the sample is contacted with an enzyme permeabilization reagent. The enzyme permeabilization reagent permeabilizes the sample by partially degrading extracellular matrix or surface proteins that prevent permeabilization of the sample by the assay reagent. Contact with the enzyme permeabilization reagent can occur at any time after fixation and before target detection. In some examples, the enzyme permeabilization reagent is the commercially available enzyme proteinase K. In such cases, the sample is contacted with proteinase K before contact with the post-fixation reagent. Proteinase K treatment (i.e., contact with proteinase K, also commonly referred to as "proteinase K digestion") can be carried out over a range of enzyme concentrations, at a range of temperatures, and for a range of times, which are empirically determined for each cell or tissue type under investigation. For example, the sample can be contacted with proteinase K for 30 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, or 2 minutes or less. The sample may be contacted with proteinase K at 1 μg / ml or less, 2 μg / ml or less, 4 μg / ml or less, 8 μg / ml or less, 10 μg / ml or less, 20 μg / ml or less, 30 μg / ml or less, 50 μg / ml or less, or 100 μg / ml or less. The sample may be contacted with proteinase K at a temperature ranging from 2°C to 55°C, with particular ranges of interest including, but not limited to, 50-54°C, 40-44°C, 35-39°C, 28-32°C, 20-26°C, and 0-6°C. In some examples, the sample may be contacted with proteinase K at a temperature of 4°C, room temperature (22-25°C), 30°C, 37°C, 42°C, or 52°C. In some embodiments, the sample is not contacted with an enzyme permeabilization reagent. In some embodiments, the sample is not contacted with proteinase K. Contacting the intact tissue with at least a fixation reagent and a permeabilization reagent results in the production of fixed and permeabilized tissue.

[0047] Ligase In some embodiments, the disclosed method includes adding a ligase to ligate a second oligonucleotide and generate a closed nucleic acid circle. In some embodiments, adding the ligase includes adding a DNA ligase. In alternative embodiments, the second oligonucleotide is provided as a closed nucleic acid circle, and the step of adding the ligase is omitted. In certain embodiments, the ligase is an enzyme that facilitates sequencing of the target nucleic acid molecule.

[0048] As used herein, the term "ligase" refers to an enzyme commonly used to join polynucleotides together or to join the ends of a single polynucleotide. Ligases include ATP-dependent double-stranded polynucleotide ligases, NAD-dependent double-stranded DNA or RNA ligases, and single-stranded polynucleotide ligases, such as any of the ligases set forth in EC 6.5.1.1 (ATP-dependent ligases), EC 6.5.1.2 (NAD+-dependent ligases), and EC 6.5.1.3 (RNA ligases). Specific examples of ligases include bacterial ligases such as E. coli DNA ligase and Taq DNA ligase, Ampligase® thermostable DNA ligase, and the like. Examples include gauze (Epicentre® Technologies Corp., part of Illumina®, Madison, Wis.), and phage ligases such as T3 DNA ligase, T4 DNA ligase, and T7 DNA ligase, and variants thereof.

[0049] Rolling Circle Amplification In some embodiments, the method of the present invention comprises performing rolling circle amplification in the presence of a nucleic acid molecule, using a second oligonucleotide as a template and a first oligonucleotide as a primer for polymerase to form one or more amplicons. In such embodiments, a single-stranded circular polynucleotide template is formed by ligation of the second oligonucleotide, and the circular polynucleotide comprises a region complementary to the first oligonucleotide. Upon addition of DNA polymerase in the presence of appropriate dNTP precursors and other cofactors, the first oligonucleotide is extended by replicating multiple copies of the template. This amplification product can be easily detected by binding to a detection probe.

[0050] In some embodiments, only if the first and second oligonucleotides hybridize to the same target nucleic acid molecule can the second oligonucleotide be circularized and subjected to rolling circle amplification to generate cDNA nanoballs (i.e., amplicons) containing multiple copies of the cDNA. The term "amplicon" refers to the amplified nucleic acid product of a PCR reaction or other nucleic acid amplification process. In some embodiments, amine-modified nucleotides are spiked into the rolling circle amplification reaction.

[0051] Techniques for rolling circle amplification are known in the art (e.g., Baner et al., Nucleic Acids Research, 26:5073-5078, 1998; Lizardi et al., Nature Genetics 19:226, 1998; Schweitzer et al. Proc. Natl. Acad. Sci. USA 97:101 13-1 19, 2000; Faruqi et al. al, BMC Genomics 2:4, 2000; Nallur et al, Nucl. Acids Res. 29:el 18, 2001; Dean et al. Genome Res. 1 1:1095-1099, 2001; Schweitzer et al, Nature Biotech. 20:359-365, 2002; U.S. Patent Nos. 6,054,274, 6,291,187, 6,323,009, 6,344,329, and 6,368,801).

[0052] In some embodiments, the polymerase is phi29 DNA polymerase. In certain aspects, the nucleic acid molecule comprises an amine-modified nucleotide. In such embodiments, the amine-modified nucleotide comprises an N-hydroxysuccinimide acrylate partial modification. Examples of other amine-modified nucleotides include, but are not limited to, a 5-aminoallyl-dUTP partial modification, a 5-propargylamino-dCTP partial modification, an N6-6-aminohexyl-dATP partial modification, or a 7-deaza-7-propargylamino-dATP partial modification.

[0053] Setting up amplicon embedding in tissue hydrogel In some embodiments, the disclosed methods include embedding one or more amplicons in the presence of hydrogel subunits to form one or more hydrogel-embedded amplicons. The described hydrogel-histochemistry involves covalently attaching nucleic acids to an in situ synthesized hydrogel for multiple cycles of tissue cleanup, enzyme diffusion, and sequencing. In some embodiments, amine-modified nucleotides are spiked into the rolling circle amplification reaction to enable the setting up of amplicon embedding in the tissue-hydrogel, and acrylic acid is used. Acid N-hydroxysuccinimide esters are used to functionalize with acrylamide moieties and copolymerize with acrylamide monomers to form hydrogels.

[0054] As used herein, the term "hydrogel" or "hydrogel network" refers to a network of water-insoluble polymer chains, sometimes found as a colloidal gel in which water is the dispersion medium. In other words, hydrogels are a class of polymeric materials that can absorb large amounts of water without dissolving. Hydrogels can contain more than 99% water and can include natural or synthetic polymers, or a combination thereof. Due to their significant water content, hydrogels also possess a degree of flexibility very similar to that of natural tissue. A detailed description of suitable hydrogels can be found in U.S. Patent Application Publication No. 2010 / 0055733, which is specifically incorporated herein by reference. As used herein, the term "hydrogel subunit" or "hydrogel precursor" refers to a hydrophilic monomer, prepolymer, or polymer that can be crosslinked or "polymerized" to form a three-dimensional (3D) hydrogel network. Without being bound by any scientific theory, it is believed that this fixation of the biological sample in the presence of the hydrogel subunits crosslinks components of the specimen to the hydrogel subunits, thereby locking the molecular components in place and preserving tissue architecture and cellular morphology.

[0055] In some embodiments, embedding comprises copolymerizing one or more amplicons with acrylamide. As used herein, the term "copolymer" describes a polymer containing two or more types of subunits. This term encompasses polymers containing two, three, four, five, or six types of subunits.

[0056] In certain aspects, embedding comprises purifying the one or more hydrogel-embedded amplicons, such that the target nucleic acid is substantially retained within the one or more hydrogel-embedded amplicons. In such embodiments, purifying comprises substantially removing cellular components from the one or more hydrogel-embedded amplicons. In some other embodiments, purifying comprises substantially removing lipids from the one or more hydrogel-embedded amplicons. As used herein, the term "substantially" means that the original amount present in the sample prior to purifying has been reduced by approximately 70% or more, such as 75% or more, such as 80% or more, such as 85% or more, such as 90% or more, such as 95% or more, such as 99% or more, or 100%.

[0057] In some embodiments, purifying the hydrogel-embedded amplicons comprises electrophoresing the specimen. In some embodiments, the amplicons are electrophoresed using a buffer solution comprising an ionic surfactant. In some embodiments, the ionic surfactant is sodium dodecyl sulfate (SDS). In some embodiments, the specimen is electrophoresed using a voltage ranging from about 10 to about 60 volts. In some embodiments, the specimen is electrophoresed for a period ranging from about 15 minutes to up to about 10 days. In some embodiments, the method further comprises incubating the purified specimen in a mounting medium having a refractive index matching the refractive index of the purified tissue. In some embodiments, the mounting medium increases the optical clarity of the specimen. In some embodiments, the mounting medium comprises glycerol.

[0058] Sequencing with error correction by dynamic annealing and ligation (SEDAL) The methods disclosed herein include contacting one or more hydrogel-embedded amplicons having a barcode sequence with a pair of primers under conditions that allow for ligation, the pair of primers including a third oligonucleotide and a fourth oligonucleotide, and ligation occurring only when both the third oligonucleotide and the fourth oligonucleotide ligate to the same amplicon. In some embodiments, the sequencing by ligation method is performed at room temperature to preserve tissue morphology, Has low background noise and reduced errors. In some embodiments, contacting one or more hydrogel-embedded amplicons includes eliminating the accumulation of errors as sequencing progresses.

[0059] In some embodiments, the contacting of one or more hydrogel-embedded amplicons occurs two or more times, including, but not limited to, three or more times, four or more times, five or more times, six or more times, or seven or more times. In certain embodiments, the contacting of one or more hydrogel-embedded amplicons occurs four or more times for thin tissue specimens. In other embodiments, the contacting of one or more hydrogel-embedded amplicons occurs six or more times for thick tissue specimens. In some embodiments, the contacting of one or more amplicons with a pair of primers may be for 24 hours or more, 24 hours or less, 18 hours or less, 12 hours or less, 8 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, 60 minutes or less, 45 minutes or less, 30 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, or 2 minutes or less.

[0060] Specimens prepared using the subject methods can be analyzed by any of several different types of microscopy, such as optical microscopy (e.g., bright field, oblique illumination, dark field, phase contrast, differential interference contrast, interference reflectance, epifluorescence, confocal microscopy, spinning disk confocal microscopy), light sheet microscopy, lattice light sheet or light field microscopy, laser microscopy, electron microscopy, and scanning probe microscopy. In some embodiments, the non-transitory computer-readable medium first converts raw images acquired via multiple rounds of in situ sequencing microscopy into decoded gene identities and spatial locations, and then analyzes the per-cell composition of gene expression.

[0061] SEDAL oligonucleotide primers In some embodiments, the disclosed method includes a third oligonucleotide and a fourth oligonucleotide. In certain aspects, the third oligonucleotide is configured to decode the base, and the fourth oligonucleotide is configured to convert the decoded base into a signal. In some aspects, the signal is a fluorescent signal. In exemplary aspects, contacting one or more hydrogel-embedded amplicons having a barcode sequence with a pair of primers under conditions that allow ligation includes ligating each of the third oligonucleotide and the fourth oligonucleotide to form a stable product for imaging only if a perfect match occurs. In certain aspects, the mismatch sensitivity of a ligase enzyme is used to determine the underlying sequence of the target nucleic acid molecule.

[0062] The term "perfectly matched" when used in reference to a double strand means that the polynucleotide and / or oligonucleotide strands constituting the double strand form a double-stranded structure with each other, so that every nucleotide in each strand undergoes Watson-Crick base pairing with the nucleotide in the other strand.The term "double strand" includes, but is not limited to, the pairing of nucleoside analogs such as deoxyinosine, nucleosides with 2-aminopurine bases, peptide nucleic acids (PNAs), etc., and these can be used.A "mismatch" in the double strand between two oligonucleotides means that a pair of nucleotides in the double strand cannot undergo Watson-Crick binding.

[0063] In some embodiments, the methods include a plurality of third oligonucleotides, including but not limited to, 5 or more third oligonucleotides, e.g., 8 or more, 10 or more, 12 or more, 15 or more, 18 or more, 20 or more, 25 or more, 30 or more, 35 or more, that hybridize to target nucleotide sequences. In some embodiments, the methods of the disclosure include 15 or more third oligonucleotides, including but not limited to, 15 or more, e.g., 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, and up to 80 different target nucleotide sequences. The method includes a plurality of third oligonucleotides, including, for example, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, and up to 80 different first oligonucleotides. In some embodiments, the method includes a plurality of fourth oligonucleotides, including, but not limited to, 5 or more fourth oligonucleotides, for example, 8 or more, 10 or more, 12 or more, 15 or more, 18 or more, 20 or more, 25 or more, 30 or more, 35 or more. In some embodiments, the method of the present disclosure comprises a plurality of fourth oligonucleotides, including but not limited to 15 or more fourth oligonucleotides, for example, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, and up to 80 different first oligonucleotides, which hybridize to 15 or more, for example, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, and up to 80 different target nucleotide sequences.Multiple oligonucleotide pairs can be used in reaction, and one or more pairs specifically bind to each target nucleic acid.For example, to improve sensitivity and reduce variability, two primer pairs can be used for one target nucleic acid. It is also of interest to detect multiple different target nucleic acids within a cell, for example, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 12, up to 15, up to 18, up to 20, up to 25, up to 30, up to 40 or more different target nucleic acids.

[0064] In certain embodiments, the SEDAL comprises a ligase whose activity is inhibited by a base mismatch, a third oligonucleotide, and a fourth oligonucleotide. The term "inhibited" in this context refers to a ligase activity that is reduced by about 20% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, such as 95% or more, such as 99% or more, or 100%. In some embodiments, the third oligonucleotide has a length of 5-15 nucleotides, including, but not limited to, 5-13 nucleotides, 5-10 nucleotides, or 5-8 nucleotides. In some embodiments, the T of the third oligonucleotide is m is room temperature (22-25°C). In some embodiments, the third oligonucleotide is denatured or partially denatured. In some embodiments, the fourth oligonucleotide has a length of 5-15 nucleotides, including but not limited to, 5-13 nucleotides, 5-10 nucleotides, or 5-8 nucleotides. In some embodiments, the T of the fourth oligonucleotide is m The temperature is room temperature (22-25°C). After each cycle of SEDAL corresponding to a base read, the fourth oligonucleotide may be stripped off, thereby eliminating error accumulation as sequencing progresses. In such an embodiment, the fourth oligonucleotide is stripped off with formamide.

[0065] In some embodiments, SEDAL involves washing the third and fourth oligonucleotides to remove unbound oligonucleotides, followed by revealing the fluorescent product for imaging. In certain exemplary embodiments, detectable labels may be used to detect one or more nucleotides and / or oligonucleotides described herein. In certain embodiments, detectable labels may be used to detect one or more amplicons. Examples of detectable markers include various radioactive moieties, enzymes, prosthetic groups, fluorescent markers, luminescent markers, bioluminescent markers, metal particles, protein-protein binding pairs, protein-antibody binding pairs, etc. Examples of fluorescent proteins include, but are not limited to, yellow fluorescent protein (YFP), green fluorescent protein (GFP), cyan fluorescent protein (CFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin, etc. Examples of bioluminescent markers include, but are not limited to, luciferases (e.g., bacterial, firefly, click beetle, etc.), luciferin, aequorin, etc. Enzymes with visually detectable signals Exemplary systems include, but are not limited to, galactosidase, glucuronidase, phosphatase, peroxidase, cholinesterase, etc. Identifiable markers include: 125 I, 35 S, 14 C, or 3 Also included are radioactive compounds such as 3H. Identifiable markers are commercially available from a variety of sources.

[0066] Fluorescent labels and their attachment to nucleotides and / or oligonucleotides are described in detail in Haugland, Handbook of Fluorescent Probes and Research Chemicals, Ninth Edition (Molecular Probes, Inc., Eugene, 2002), Keller and Manak, DNA Probes, 2nd Edition (Stockton, NY, 2003), and in Molecular Probes, 3rd Edition (Molecular Probes, Inc., Eugene, 2003). Press, New York, 1993), Eckstein, editor, Oligonucleotides and Analogues: A Practical The methods are described in many reviews, including "The Approach" (IRL Press, Oxford, 1991) and Wetmur, Critical Reviews in Biochemistry and Molecular Biology, 26:227-259 (1991). Specific methodologies applicable to the present invention are disclosed in references U.S. Patent Nos. 4,757,141, 5,151,507, and 5,091,519. In one embodiment, one or more fluorescent dyes are used as labels for labeled target sequences, as disclosed, for example, by U.S. Pat. No. 5,188,934 (4,7-dichlorofluorescein dyes), U.S. Pat. No. 5,366,860 (spectrally resolvable rhodamine dyes), U.S. Pat. No. 5,847,162 (4,7-dichlororhodamine dyes), U.S. Pat. No. 4,318,846 (ether-substituted fluorescein dyes), U.S. Pat. No. 5,800,996 (energy transfer dyes), and Li et al., U.S. Pat. No. 5,066,580 (xanthine dyes), U.S. Pat. No. 5,688,648 (energy transfer dyes), etc. Labeling can also be performed using quantum dots, as disclosed in the following patents and patent publications: U.S. Patent Nos. 6,322,901; 6,576,291; 6,423,551; 6,251,303; 6,319,426; 6,426,513; 6,444,143; 5,990,479; 6,207,392; 2002 / 0045045; and 2003 / 0017264. As used herein, the term "fluorescent label" includes signaling moieties that communicate information via the fluorescent absorption and / or emission properties of one or more molecules. Such fluorescent properties include fluorescence intensity, fluorescence lifetime, emission spectral characteristics, energy transfer, and the like.

[0067] Commercially available fluorescent nucleotide analogs that are readily incorporated into nucleotide and / or oligonucleotide sequences include Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dUTP (Amersham Biosciences, Piscataway, NJ), fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, TEXAS RED™-5-dUTP, CASCADE BLUE™-7-dUTP, BODIPY TMFL-14-dUTP, BODIPY TMR-14-dUTP, BODIPY TMTR-14-dUTP, RHODAMINE GREEN™-5-dUTP, OREGON GREEN™ 488-5 ... TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED RED(TM)-12-dUTP, BODIPY(TM) 630 / 650-14-dUTP, BODIPY(TM) 650 / 665-14-dUTP, ALEXA FLUOR(TM) 488-5-dUTP, ALEXA FLUOR(TM) 532-5-dUTP, ALEXA FLUOR(TM) 568-5-dUTP, ALEXA FLUOR(TM) 594-5-dUTP, ALEXA FLUOR(TM) 546-14-dUTP, Fluorescein-12-UTP, Tetramethylrhodamine-6-UTP, TEXAS RED(TM)-5-UTP, mCherry, CASCADE BLUE(TM)-7-UTP, BODIPY(TM) FL-14-UTP, BODIPY TM Examples of suitable fluorophore-containing nucleotides include, but are not limited to, R-14-UTP, BODIPY™ TR-14-UTP, RHODAMINE GREEN™-5-UTP, ALEXA FLUOR™ 488-5-UTP, LEXA FLUOR™ 546-14-UTP (Molecular Probes, Inc. Eugene, Oreg.), etc. Protocols for custom synthesis of nucleotides bearing other fluorophores are known in the art (see Henegariu et al. (2000) Nature Biotechnol. 18:345).

[0068] Other fluorophores available for post-synthetic conjugation include ALEXA FLUOR™ 350, ALEXA FLUOR™ 532, ALEXA FLUOR™ 546, ALEXA FLUOR™ 568, ALEXA FLUOR™ 594, ALEXA FLUOR™ 647, BODIPY 493 / 503, BODIPY FL, BODIPY R6G, BODIPY 530 / 550, BODIPY TMR, BODIPY 558 / 568, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, Cascade Blue, Cascade Yellow, dansyl, Lissamine Rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pathic Blue, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Tetramethylrhodamine, Texas Red (available from Molecular Probes, Inc., Eugene, Oregon), Cy2, Cy3.5, Cy5.5, Cy7 (Amersham Biosciences, Piscataway, NJ), etc. FRET tandem fluorophores can also be used, including, but not limited to, PerCP-Cy5.5, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, APC-Cy7, PE-Alexa dyes (610, 647, 680), APC-Alexa dyes, etc.

[0069] Metallic silver or gold particles can be used to enhance the signal from fluorescently labeled nucleotide and / or oligonucleotide sequences (Lakowicz et al. (2003) BioTechniques 34:62).

[0070] Biotin or its derivatives can be used as a label on nucleotide and / or oligonucleotide sequences, followed by binding with a detectably labeled avidin / streptavidin derivative (e.g., phycoerythrin-conjugated streptavidin) or a detectably labeled anti-biotin antibody. Digoxigenin can be incorporated as a label, followed by binding with a detectably labeled anti-digoxigenin antibody (e.g., fluoresceinated anti-digoxigenin). Aminoallyl-dUTP residues can be incorporated into oligonucleotide sequences, followed by binding to N-hydroxysuccinimide (NHS)-derivatized fluorescent dyes. Generally, any member of a conjugate pair can be incorporated into a detection oligonucleotide, provided that the detectably labeled conjugate partner can be conjugated in a manner that allows detection. As used herein, the term "antibody" refers to any class of antibody molecule, or any subfragment thereof, such as Fab.

[0071] Other suitable labels for oligonucleotide sequences may include fluorescein (FAM), digoxigenin, dinitrophenol (DNP), dansyl, biotin, bromodeoxyuridine (BrdU), hexahistidine (6xHis), phosphorescent amino acids (e.g., P-tyr, P-ser, P-thr), etc. In one embodiment, the following hapten / antibody pairs are used for detection, with each of the antibodies derivatized with a detectable label: biotin / α-biotin, digoxigenin / α-digoxigenin, dinitrophenol (DNP) / α-DNP, 5-carboxyfluorescein (FAM) / α-FAM.

[0072] In certain exemplary embodiments, nucleotide and / or oligonucleotide sequences can be indirectly labeled with a hapten that is then bound by a capture agent, as disclosed, for example, in U.S. Pat. Nos. 5,344,757, 5,702,888, 5,354,657, 5,198,537, and 4,849,336, PCT Publication No. 91 / 17160, etc. Many different hapten-capture agent pairs can be used. Exemplary haptens include, but are not limited to, biotin, desbiotin and other derivatives, dinitrophenol, dansyl, fluorescein, CY5, digoxigenin, etc. In the case of biotin, the capture agent can be avidin, streptavidin, or an antibody. Antibodies can be used as capture agents for other haptens (many dye-antibody pairs are commercially available, e.g., Molecular Probes, Eugene, Oreg.).

[0073] cell The methods disclosed herein include methods for in situ gene sequencing of target nucleic acids in cells in intact tissues. In certain embodiments, the cells are present in a population of cells. In certain other embodiments, the population of cells comprises multiple cell types, including, but not limited to, excitatory neurons, inhibitory neurons, and non-neuronal cells. The cells used in the assays of the present invention can be from an organism, a single cell type derived from an organism, or a mixture of cell types. Naturally occurring cells and cell populations, genetically engineered cell lines, cells derived from transgenic animals, and the like, can be included. Virtually any cell type and size can be accommodated. Suitable cells include bacterial, fungal, plant, and animal cells. In one embodiment of the present invention, the cells are mammalian cells, e.g., complex cell populations from naturally occurring tissues, such as blood, liver, pancreas, nervous tissue, bone marrow, and skin. Some tissues can be disrupted into monodisperse suspensions. Alternatively, the cells can be cultured populations, e.g., cultures derived from complex populations, cultures derived from a single cell type in which cells have differentiated into multiple lineages or in which cells respond differentially to stimuli, and the like.

[0074] Cell types that may find use in the subject invention include stem and progenitor cells, e.g., embryonic stem cells, hematopoietic stem cells, mesenchymal stem cells, neural crest cells, etc.; endothelial cells; myocytes; cardiac muscle cells; smooth and skeletal muscle cells; mesenchymal cells; epithelial cells; hematopoietic cells, such as lymphocytes, including T cells, such as Th1 T cells, Th2 T cells, ThOT cells, and cytotoxic T cells; B cells; pre-B cells; monocytes; dendritic cells; neutrophils; and macrophages; natural killer cells; mast cells; adipocytes; cells involved in specific organs, such as the thymus, endocrine glands, pancreas, and brain, such as neurons, glia, astrocytes, and dendritic cells; and genetically modified versions thereof. Hematopoietic cells may be involved in inflammatory processes, autoimmune diseases, etc., while endothelial cells, smooth muscle cells, cardiac muscle cells, etc., may be associated with cardiovascular diseases. Almost any type of cell may be involved in tumors, such as sarcomas, carcinomas, and lymphomas; liver diseases involving hepatocytes; kidney diseases involving renal cells; etc.

[0075] The cells may also be transformed or tumor cells of different types, e.g., carcinomas of different cellular origin, lymphomas of different cell types, etc. The American Type Culture Collection (Manassas, VA) has collected and made available over 950 cancer cell lines, including over 4,000 cell lines from over 150 different species and 700 human cancer cell lines. The National Cancer Institute compiles clinical, biochemical, and molecular data from a large panel of human tumor cell lines, which are available from the ATCC or NCI (Phelps et al., 2004). al. (1996) Journal of Cellular Biochemistry Supplement 24:32-91). Different cell lines may be spontaneously derived or selected from individual cell lines for desired growth or response characteristics, and may include multiple cell lines derived from similar tumor types but from different patients or sites.

[0076] Cells may be non-adherent, e.g., blood cells including monocytes, T cells, B cells, tumor cells, etc., or adherent cells, e.g., epithelial cells, endothelial cells, neuronal cells, etc. To profile adherent cells, they may be dissociated from the substrate to which they are adhered and from other cells in a manner that maintains their ability to recognize and bind to probe molecules.

[0077] Such cells can be obtained from an individual, for example, by various techniques known in the art, from various tissues, e.g., blood, bone marrow, solid tissue (e.g., solid tumors), ascites, using extraction, lavage, wash, surgical dissection, etc. Cells can be obtained from fixed or unfixed, fresh or frozen, whole or disaggregated samples. Disaggregation of tissue can occur either mechanically or enzymatically using known techniques.

[0078] Imaging The disclosed methods include imaging one or more hydrogel-embedded amplicons using any of several different types of microscopy, such as confocal microscopy, two-photon microscopy, light field microscopy, intact tissue expansion microscopy, and / or CLARITY™ optimized light sheet microscopy (COLM).

[0079] Bright-field microscopy is the simplest of all optical microscopy techniques. Sample illumination is via transmitted white light, i.e., illuminated from below and observed from above. Limitations include the low contrast of most biological samples and the poor apparent resolution due to blurring of out-of-focus material. The simplicity of the technique and the minimal sample preparation required are significant advantages.

[0080] In oblique illumination microscopy, the specimen is illuminated from the side. This gives the image a three-dimensional appearance and can highlight features that would otherwise be invisible. A more recent technique based on this method is Hoffman modulation contrast, a system found in inverted microscopes used for cell culture. Oblique illumination suffers from the same limitations as brightfield microscopy (low contrast in many biological specimens, apparent poor resolution due to out-of-focus objects), but can highlight structures that would otherwise be invisible.

[0081] Dark-field microscopy is a technique for improving the contrast of unstained, transparent specimens. Dark-field illumination uses a carefully aligned light source to minimize the amount of directly transmitted (unscattered) light entering the image plane and collect only the light scattered by the specimen. Dark-field illumination can dramatically improve image contrast (especially of transparent objects) with little instrument setup and specimen preparation required. However, this technique continues to suffer from low light intensity in the final image for many biological specimens and suffers from poor apparent resolution.

[0082] Phase contrast is an optical microscopy illumination technique that converts the phase shift of light passing through a transparent specimen into a change in image brightness. That is, phase contrast reveals differences in refractive index as differences in contrast. The phase shift itself is invisible to the human eye, but becomes apparent as the brightness changes.

[0083] In differential interference contrast (DIC) microscopy, differences in optical density are shown as differences in relief. The system consists of a special prism (Nomarski prism, Wollaston prism) in a condenser, which splits the light into ordinary and extraordinary beams. The spatial difference between the two beams is minimal (less than the maximum resolution of the objective). After passing through the specimen, the beams are recombined by a similar prism in the objective. In a homogeneous specimen, 2 There is no difference between the two beams, and no contrast is produced. However, near a refractive boundary (e.g., the nucleus within the cytoplasm), the difference between the ordinary and extraordinary beams creates relaxation in the image. Differential interference contrast requires a polarized light source to function, and two polarizing filters must be installed in the light path: one below the condenser (polarizer) and one above the objective lens (analyzer).

[0084] Another microscopy technique that uses interference is interference reflectance microscopy (also known as reflection interference contrast, or RIC). It is used to study cell adhesion to glass surfaces using polarized light over a narrow range of wavelengths that is reflected whenever there is an interface between two materials with different refractive indices. When cells bind to the glass surface, the light reflected from the glass interferes with the light reflected from the bound cells. When there are no cells bound to the glass, there is no interference.

[0085] A fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence instead of, or in addition to, reflection and absorption to study the properties of organic or inorganic materials. In fluorescence microscopy, a sample is illuminated with light of a wavelength that excites fluorescence in the sample. The fluorescent light, usually at a longer wavelength than the illumination, is then imaged through a microscope objective. This technique can use two filters: an illumination (or excitation) filter, which ensures that the illumination is near monochromatic and of the correct wavelength, and a second emission (or barrier) filter, which ensures that none of the excitation light source reaches the detector. Alternatively, both of these functions can be achieved by a single dichroic filter. "Fluorescence microscope" refers to any microscope that uses fluorescence to produce images, whether it is a simpler setup like a fluorescent microscope or a more complex design like a confocal microscope, which uses optical sectioning to obtain better resolution of the fluorescent image.

[0086] In some embodiments, antioxidant compounds are included in the wash and imaging buffers (i.e., "anti-fade buffers") to reduce photobleaching during fluorescence imaging. Exemplary antioxidants include, but are not limited to, propyl gallate, tertiary butylhydroquinone, butylated hydroxyanisole, butylated hydroxytoluene, glutathione, ascorbic acid, and tocopherol. Such antioxidants have an anti-fade effect on fluorophores. That is, antioxidants reduce photobleaching during tiling and significantly enhance the SNR of sensitive fluorophores, allowing for higher SNR imaging of thicker samples. For fixed exposure times, antioxidants increase the SNR by increasing the concentration of unbleached fluorophores during exposure to light. Including antioxidants also eliminates the diminishing returns of longer exposure times (caused by limited fluorescence lifetime before photobleaching), providing increased SNR by allowing for longer exposure times.

[0087] Additionally, fluorophore cleavage from the probe (as opposed to probe detachment) is used in the CISITS method to eliminate round-to-round signal. The inclusion of a disulfide bond between the fluorophore and the oligonucleotide probe allows for fluorophore cleavage from the oligonucleotide probe in a reducing environment. After fluorescent imaging of a sequencing round, a reducing agent is added to reduce the disulfide bond connecting the fluorophore to the oligonucleotide, and a subsequent washing step removes the diffusible fluorescent signal before performing another round. The use of SEDAL sequencing with thiol-based chemical cleavage in CISITS combines the specificity and SNR advantages of SEDAL sequencing with the robustness and rapidity of round-to-round cycling from fluorophore cleavage.

[0088] Confocal microscopes use point illumination and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus signals. They only detect light produced by fluorescence very close to the focal plane. Because the pinhole cannot capture light, the optical resolution of images, especially across the sample depth, is far superior to that of wide-field microscopes. However, this increased resolution comes at the expense of reduced signal intensity, often necessitating long exposure times, as much of the light from the sample fluorescence is blocked by the pinhole. Because only one point on the sample is illuminated at a time, 2D or 3D imaging requires scanning across a regular raster (i.e., a rectangular pattern of parallel scan lines) across the specimen. The achievable thickness of the focal plane is primarily defined by the wavelength of light used divided by the numerical aperture of the objective lens, but also by the optical properties of the specimen. Thin optical sectioning makes these types of microscopes particularly excellent for 3D imaging and surface profiling of samples. COLM offers an alternative microscope for high-speed 3D imaging of large, well-defined samples. COLM allows for the examination of large tissues, including immunoassays, and allows for increased collection speeds, resulting in higher quality data.

[0089] In single-plane illumination microscopy (SPIM), also known as light-sheet microscopy, only fluorophores in the focal plane of the detection objective are illuminated. The light sheet is a beam collimated in one direction and focused in the other. Because fluorophores outside the focal plane of the detector are not excited, the method also provides unique optical sectioning. Furthermore, compared to traditional microscopy, light-sheet methods exhibit reduced photobleaching and phototoxicity, often allowing for many more scans per specimen. By rotating the specimen, the technique can image virtually any plane with multiple views obtained from different angles. However, for all angles, only relatively shallow sections of the specimen are imaged with high resolution, while deeper regions appear increasingly blurred.

[0090] Super-resolution microscopy is a form of optical microscopy. Due to the diffraction of light, the resolution of conventional light microscopy is limited, as stated by Ernst Abbe in 1873. A good approximation of the achievable resolution is the FWHM (full width at half maximum) of the point spread function; precision wide-field microscopes with high apertures and visible light typically reach a resolution of approximately 250 nm. Super-resolution techniques allow images to be captured at resolutions higher than the diffraction limit. These fall into two broad categories: "true" super-resolution techniques, which capture the information contained in evanescent waves, and "functional" super-resolution techniques, which use experimental techniques and known limitations of the material being imaged to reconstruct super-resolution images.

[0091] Laser microscopy uses laser illumination sources in various forms of microscopy. For example, laser microscopy focused on biological applications uses ultrashort pulsed lasers, or femtosecond lasers, in several techniques, including nonlinear microscopy, saturation microscopy, and multiphoton fluorescence microscopy such as two-photon excitation microscopy (a fluorescence imaging method that allows imaging of living tissues to very deep depths, e.g., up to 1 mm).

[0092] Electron microscopes (EM) use a beam of electrons to illuminate a specimen, producing a magnified image. Electron microscopes have a wavelength approximately 100,000 times shorter than visible light (photons), giving them higher resolution than photoelectric optical microscopes. They can achieve resolutions better than 50 pm and magnifications up to approximately 10,000,000 times, whereas conventional non-focusing optical microscopes are limited by diffraction to a resolution of approximately 200 nm and a useful magnification of less than 2000 times. Electron microscopes use electrostatic and electromagnetic "lenses" to control and focus the electron beam to form an image. These lenses are similar to, but different from, the glass lenses of a light microscope, which form a magnified image by focusing light on or through the specimen. Electron microscopes are used to observe a wide range of biological and inorganic specimens, including microorganisms, cells, large molecules, biopsies, metals, and crystals. In industry, electron microscopes are often used for quality control and failure analysis. Examples of electron microscopy include transmission electron microscopy (TEM), scanning electron microscopy (SEM), and reflection electron microscopy (REM). , scanning transmission electron microscopy (STEM) and low voltage electron microscopy (LVEM).

[0093] Scanning probe microscopy (SPM) is a branch of microscopy that uses a physical probe to scan a specimen to form an image of a surface. The image of the surface is obtained by mechanically moving the probe line-by-line in a raster scan of the specimen and recording the interaction of the probe with the surface as a function of position. Examples of SPM include atomic force microscopy (ATM), ballistic electron emission microscopy (BEEM), chemical force microscopy (CFM), conductive atomic force microscopy (C-AFM), electrochemical scanning tunneling microscopy (ECSTM), electrostatic force microscopy (EFM), fluid force microscopy (FluidFM), force modulation microscopy (FMM), feature-oriented scanning probe microscopy (FOSPM), Kelvin probe force microscopy (KPFM), magnetic force microscopy (MFM), magnetic resonance force microscopy (MRFM), near-field scanning optical microscopy (NSOM) (or SNOM, scanning near-field optical microscope, SNOM, piezoresponse force microscopy (PFM), PSTM, photon scanning tunneling microscopy (PSTM), PTMS, and photothermal microscopy. Spectroscopy / Microscopy (PTMS), SCM, Scanning Capacitance Microscopy (SCM), SECM, Scanning Electrochemical Microscopy (SECM), SGM, Scanning Gate Microscopy (SGM), SHPM, Scanning Hall Probe Microscopy (SHPM), SICM, Scanning Ion Conductance Microscopy (SICM), SPSM Spin Polarized Scanning Tunneling Microscopy (SPSM), SSRM, Scanning Spreading Resistance Microscopy (SSRM), SThM, Scanning Thermal Microscopy (SThM), STM, Scanning Tunneling Microscopy (STM), STP, Scanning Tunneling Potentiometry (STP), SVM, Scanning Voltage Microscopy (SVM), and Synchrotron X-ray Scanning Tunneling Microscopy (SXSTM).

[0094] Intact tissue expansion microscopy (exM) allows imaging of thick, preserved specimens with a lateral resolution of approximately 70 nm. Using ExM, the optical diffraction limit is circumvented by physically expanding the biological specimen before imaging, bringing sub-diffraction-limited structures into the size range visible by conventional diffraction-limited microscopes. ExM can image biological specimens at the voxel ratio of diffraction-limited microscopes but at the voxel size of super-resolution microscopes. Expanded specimens are transparent, and the expanded material is >99% water, making it index-matched to water. Expansion microscopy techniques are known in the art, as disclosed, for example, in Gao et al., Q&A: Expansion Microscopy, BMC Biol. 2017;15:50.

[0095] Screening Methods The methods disclosed herein also provide a method of screening a candidate agent to determine whether the candidate agent modulates gene expression of a nucleic acid in cells in an intact tissue, the method comprising: (a) contacting a fixed and permeabilized intact tissue sample with at least a pair of oligonucleotide primers under conditions that allow for specific hybridization, the pair of primers comprising a first oligonucleotide and a second oligonucleotide, each of the first oligonucleotide and the second oligonucleotide comprising a first region of complementarity, a second region of complementarity, and a third region of complementarity, the second oligonucleotide further comprising a barcode sequence, the first region of complementarity of the first oligonucleotide being complementary to a first portion of a target nucleic acid; (b) contacting two oligonucleotides, each of which has a second complementary region of the first oligonucleotide complementary to a first complementary region of the second oligonucleotide, a third complementary region of the first oligonucleotide complementary to a third complementary region of the second oligonucleotide, the second complementary region of the second oligonucleotide complementary to a second portion of the target nucleic acid, and the first complementary region of the first oligonucleotide adjacent to the second complementary region of the second oligonucleotide; (b) adding a ligase to ligate the second oligonucleotides to form a closed nucleic acid circle; and (c) performing rolling circle amplification in the presence of the nucleic acid molecule, using the second oligonucleotide as a template and the first oligonucleotide as a primer for a polymerase. (d) embedding the one or more amplicons in the presence of hydrogel subunits to form one or more hydrogel-embedded amplicons; (e) contacting the one or more hydrogel-embedded amplicons having the barcode sequence with a pair of primers under conditions that allow ligation, wherein the pair of primers comprises a third oligonucleotide and a fourth oligonucleotide, and ligation occurs only if both the third oligonucleotide and the fourth oligonucleotide ligate to the same amplicon; (f) repeating step (e); (g) imaging the one or more hydrogel-embedded amplicons to determine in situ gene sequencing of the target nucleic acid in cells in the intact tissue; and (h) detecting a gene expression level of the target nucleic acid, wherein a change in the expression level of the target nucleic acid in the presence of the at least one candidate agent relative to the expression level of the target nucleic acid in the absence of the at least one candidate agent indicates that the at least one candidate agent modulates gene expression of the nucleic acid in cells in the intact tissue. Such screening methods include the steps of CISITS provided herein.

[0096] In some embodiments, the detection comprises performing flow cytometry, sequencing, probe binding and electrochemical detection, pH change, catalysis induced by enzymes bound to DNA tags, quantum entanglement, Raman spectroscopy, terahertz wave technology, and / or scanning electron microscopy. In certain embodiments, the flow cytometry is mass cytometry or fluorescence-activated flow cytometry. In some other embodiments, the detection comprises performing microscopy, scanning mass spectrometry, or other imaging techniques described herein. In such embodiments, the detection comprises determining a signal, for example, a fluorescent signal.

[0097] As used interchangeably herein, the terms "test agent," "candidate agent," and grammatical equivalents refer to any molecule (e.g., a protein (herein including proteins, polypeptides, and peptides), a small molecule (i.e., between 5 and 1000 Da, 100 and 750 Da, 200 and 500 Da, or less than 500 Da in size), or an organic or inorganic molecule, polysaccharide, polynucleotide, etc.) that is tested for activity in the subject assay.

[0098] A variety of different candidate agents can be screened by the above methods. Candidate agents encompass numerous chemical classes, such as small organic compounds having molecular weights of more than 50 daltons (e.g., at least about 50 Da, at least about 100 Da, at least about 150 Da, at least about 200 Da, at least about 250 Da, or at least about 500 Da), and less than about 20,000 daltons, less than about 10,000 daltons, less than about 5,000 daltons, or less than about 2,500 daltons. For example, in some embodiments, suitable candidate agents are organic compounds having a molecular weight in the range of about 500 Da to about 20,000 Da, e.g., about 500 Da to about 1000 Da, about 1000 Da to about 2000 Da, about 2000 Da to about 2500 Da, about 2500 Da to about 5000 Da, about 5000 Da to about 10,000 Da, or about 10,000 Da to about 20,000 Da.

[0099] Candidate agents may contain functional groups necessary for structural interaction with proteins, such as hydrogen bonding, and may include at least two of an amine, carbonyl, hydroxyl, or carboxyl group, or functional chemical groups. Candidate agents may include cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof.

[0100] Candidate agents can be obtained from a wide variety of sources, including libraries of synthetic or natural compounds. Numerous means are available for the random and directed synthesis of a wide variety of organic compounds and biomolecules, including, for example, expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds can be readily modified through conventional chemical, physical, and biochemical means and used to generate combinatorial libraries. Known pharmacological agents can undergo directed or random chemical modifications, such as acylation, alkylation, esterification, amidation, etc., to generate structural analogs. Furthermore, screening can target known pharmacologically active compounds and their chemical analogs, or novel agents with unknown properties, such as those created through rational drug design.

[0101] In one embodiment, candidate modulator is synthetic compound.Any number of methods are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including the expression of randomized oligonucleotides and oligopeptides.For example, see WO94 / 24314, which is expressly incorporated herein by reference, and discusses the method of generating new compounds, including random chemical methods and enzymatic methods.

[0102] In another embodiment, candidate agents are provided as libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts that are available or readily produced. Additionally, natural or synthetically produced libraries and compounds can be readily modified through conventional chemical, physical, and biochemical means. Known pharmacological agents can be subjected to directed or random chemical modification, including enzymatic modification, to produce structural analogs.

[0103] In one embodiment, candidate agents include proteins (antibodies, antibody fragments (i.e., fragments containing antigen binding regions, single chain antibodies, etc.), nucleic acids, and chemical moieties. In one embodiment, candidate agents are naturally occurring proteins or fragments of naturally occurring proteins. Thus, for example, cellular extracts containing the protein, or randomized or directed digests of proteinaceous cellular extracts, may be tested. In this manner, libraries of prokaryotic and eukaryotic proteins may be generated for screening. Other embodiments include libraries of bacterial, fungal, viral, and mammalian proteins (e.g., human proteins).

[0104] In one embodiment, the candidate agent is an organic moiety. In this embodiment, the candidate agent is synthesized from a series of substrates that can be chemically modified, generally as described in WO 94 / 24314. As used herein, "chemical modification" includes conventional chemical and enzymatic reactions. These substrates generally include, but are not limited to, alkyl groups (including alkanes, alkenes, alkynes, and heteroalkyls), aryl groups (including arenes and heteroaryls), alcohols, ethers, amines, aldehydes, ketones, acids, esters, amides, cyclic compounds, heterocyclic compounds (including purines, pyrimidines, benzodiazepines, beta-lactams, tetracyline, cephalosporins, and carbohydrates), steroids (including estrogens, androgens, cortisone, ecodysone, etc.), alkaloids (including ergots, vincas, curares, pyrrolizidines, and mitomycins), organometallic compounds, heteroatom-bearing compounds, amino acids, and nucleosides. Chemical (including enzymatic) reactions can be performed on moieties to form new substrates or candidate drugs, which can then be tested using the present invention.

[0105] Devices and Systems Also included are devices for practicing aspects of the CISITS method. The subject devices may be, for example, sample chambers, imaging chambers, electrophoresis apparatus, flow chambers, microscopes, needles, tubes, or the like. The pump may include a tube, a pump, and a pump.

[0106] The present disclosure also provides systems for carrying out the subject methods. The systems may include one or more of the modules described herein, such as a power supply, a refrigeration unit, a waste disposal unit, a heating unit, a pump, etc. The systems may also include any of the reagents described herein, such as an imaging buffer, a washing buffer, a stripping buffer, Nissl and DAPI solutions. Systems according to certain embodiments may also include a microscope and / or associated imaging devices, such as a camera component, a digital imaging component and / or an image capture device, a computer processor configured to collect images according to one or more user inputs, etc.

[0107] An exemplary fluidic system for implementing the CISITS method described herein is shown in Figure 7. The system described herein includes a fluidic device having a non-pressurized sample chamber with a lid having an interface tube, a first end of the interface tube held by the lid, and a second end of the interface tube positioned above the tissue sample in the sample chamber. In some embodiments, the sample chamber is a well of a multiwell plate (e.g., a 6-, 12-, or 24-well plate) or a slide chamber. The interface tube is preferably positioned sufficiently close to the bottom edge of the sample chamber so that liquid in the sample chamber remains in contact with the bottom of the interface tube as long as the liquid remains in the sample chamber. Sufficient space is left between the bottom of the sample interface tube and the sample chamber so that liquid added to the sample does not pressurize the interface tube but instead spreads and immerses the sample. In some embodiments, the interface tube is positioned at a polar angle relative to the tissue sample plane. A fluid line is connected to the first end of the interface tube so that fluid flows through the fluid line into the interface tube and out the second end of the interface tube onto the tissue sample in the sample chamber. The fluidic system may include a cryostat to maintain the sample chamber at a cryogenic temperature. The fluidic system also includes an imaging system, a pump, and a processor unit configured to perform the CISITS methods described herein.

[0108] In some embodiments, the system allows for automation of the process described herein, CISITS, which includes, but is not limited to, repeated rounds of hybridization of probes to gel-embedded DNA, ligation of fluorescently labeled oligonucleotides onto these probes, washing away excess probe, imaging, and cleavage of the fluorophore from the probe before performing the next round of sequencing. In some embodiments, the system may allow for continuous operation. In some embodiments, the system includes an imaging chamber for flowing sequencing chemicals involved in in situ DNA sequencing of the sample. In some embodiments, a fluidics and pump system controls the delivery of sequencing chemicals to the sample.

[0109] Buffers may be added / removed / recirculated / exchanged through the use of one or more ports, and optionally tubing, pumps, valves, or any other suitable fluid handling and / or fluid manipulation apparatus, e.g., tubing removably or permanently attached to one or more components of the device. For example, a first tube having a first and second end may be attached to the first port, and a second tube having a first and second end may be attached to the second port, the first end of the first tube attached to the first port and the second end of the first tube operably connected to a container, e.g., a cooling unit, a heating unit, a filtration unit, a waste container, etc., the first end of the second tube attached to the second port and the second end of the second tube operably connected to a container, e.g., a cooling unit, an ice beaker, a filtration unit, a waste container, etc. is linked to.

[0110] In some embodiments, the multi-way selector valve is interfaced with a pump. The multi-way selector valve can be used to switch currently open fluid lines to enable various modes of operation. The pump can be used to draw or push a volume of fluid. For example, a syringe pump can be used to draw a desired amount of reagent into the pump through a series of selector valves. In some embodiments, the multi-port selector valve is coupled to a reagent or buffer tray to allow the pump to draw a selected reagent or buffer into the system. The fluidic system may further include an adjustable stage supporting the reagent tray and a position sensor that enables positioning of the adjustable stage to enable selection of a desired reagent from the reagent tray fluidly connected to the multi-way selector valve. A chiller may be included in the fluidic system to maintain a desired temperature of the reagent. The pump can also be used to draw reagent from the sample well through the interface tubing. In addition, the pump can be used to push a volume through the interface tubing, for example, to the sample well or to a waste container. Alternatively, the pump can push a volume back into a reagent bank (which may be used for purging purposes).

[0111] When performing the CISITS method, it is preferable to use fresh reagents, particularly for SEDAL sequencing, rolling circle amplification, and ligation. In some embodiments, disposable reagent trays containing reagents for performing the CISITS method are used in the fluidic system, allowing for the convenient use of fresh reagents for the user.

[0112] In some embodiments, the system further includes a means for cryosectioning the tissue sample and placing the tissue section flat on a surface within the sample chamber. For example, the sample chamber (e.g., a well of a multi-well plate or slide chamber) can be kept at a low temperature in a cryostat along with a thin-walled metal ring. After the section is collected, the metal ring is pressed down onto the section so that the edge of the ring is in even contact with the cryosectioning medium surrounding the section. The ring and flat section can then be transferred to the sample chamber. The metal ring facilitates placing the section flat on a surface within the sample chamber, and the warmth of a finger can be used to separate the section from the ring and adhere it to the sample chamber surface.

[0113] In some embodiments, the system is enclosed in an enclosure that blocks ambient light. The enclosure can include several doors and drawers for accessing various internal components of the system. For example, the system may include a drawer for loading and removing samples from the system and a drawer for loading and removing reagents from the system. Other components of the enclosure may provide access for service professionals to modify, upgrade, or repair internal parts of the system. The enclosure may also be sound-attenuating.

[0114] In some embodiments, the system includes a non-transitory computer-readable storage medium having instructions that, when executed by the processor unit, cause the processor unit to control the delivery of chemicals and synchronize this process with the microscope. In some embodiments, the non-transitory computer-readable storage medium includes instructions that, when executed by the processor unit, cause the processor unit to measure the optical signal.

[0115] usefulness The devices, methods, and systems herein find several uses in arts such as biomedical research and / or clinical diagnostics. For example, in biomedical research, applications include spatially resolved imaging of cells for basic biology or drug screening. This includes, but is not limited to, gene expression analysis. In clinical diagnostics, applications include, but are not limited to, detecting genetic markers such as disease, immune response, bacterial or viral DNA / RNA in patient samples. Examples of advantages of the methods described herein include efficiency, taking only 3 or 4 days to obtain final data from raw samples, providing speeds much faster than existing microarray or sequencing technologies, being highly multiplexed (up to 1000 genes), single-cell and single-molecule sensitivity, preserved tissue morphology, and / or a high signal-to-noise ratio with a low error rate.

[0116] In certain aspects, CISITS can be applied to study molecularly defined cell types and activity-regulated gene expression in the mouse visual cortex, and can be extended to larger 3D tissue blocks to visualize short- and long-range spatial organization of cortical neurons at volumetric scales previously inaccessible. In some embodiments, the methods disclosed herein can be adapted to image DNA-conjugated antibodies for highly multiplexed protein detection.

[0117] The devices, methods, and systems of the present invention can be generalized to study several heterogeneous cell populations in diverse tissues. Without being bound by any scientific theory, the brain poses special challenges that are highly amenable to CISITS analysis. For example, polymorphic activity-regulated gene (ARG) expression observed across different cell types likely depends on both intrinsic cell biological properties (e.g., signaling pathway component expression) and external properties, such as the anatomy of neural circuits that route external sensory information to different cells (here, the visual cortex). In such cases, in situ transcriptomics, as exemplified by CISITS, can effectively link imaging-based molecular information with anatomical and activity information, thus elucidating brain function and dysfunction.

[0118] The devices, methods, and systems disclosed herein allow for the removal of cellular components, such as lipids, that typically provide structural support but prevent visualization of intracellular proteins and molecules while maintaining the three-dimensional architecture of cells and tissues, as the sample is crosslinked to a hydrogel that physically supports the tissue ultrastructure. This removal renders the interior of the biological specimen substantially transparent to light and / or macromolecules, allowing the interior of the specimen, e.g., cells and subcellular structures, to be visualized microscopically without time-consuming and destructive sectioning of the tissue. This procedure is also faster than procedures commonly used in the art, as clearing and permeabilization, typically performed in separate steps, can be combined into a single step that removes cellular components. Additionally, specimens can be repeatedly stained, unstained, and restained with other reagents for comprehensive analysis. Further functionalization with polymerizable acrylamide moieties allows amplicons to be covalently immobilized within the polyacrylamide network at multiple sites.

[0119] In one example, the subject devices, methods, and systems can be used to evaluate, diagnose, or monitor disease. As used herein, "diagnosis" generally includes predicting a subject's susceptibility to a disease or disorder, determining whether a subject is currently affected by a disease or disorder, predicting whether a subject will be affected by a disease or disorder (e.g., identifying the cancer state, stage of cancer, or the likelihood that a patient will die from cancer), predicting a subject's responsiveness to treatment of a disease or disorder (e.g., positive response, negative response, or no response at all to, for example, allogeneic hematopoietic stem cell transplantation, chemotherapy, radiation therapy, antibody therapy, or small molecule compound therapy), and using a therapy (e.g., monitoring a subject's condition and providing information on the effectiveness or efficacy of a therapy). For example, a biopsy can be prepared from cancer tissue and microscopically analyzed to determine the type of cancer, the extent to which the cancer has developed, whether the cancer will respond to a therapeutic intervention, etc.

[0120] The subject devices, methods, and systems also include methods for assessing their effect on tissue or disease. The present invention provides a useful technique for screening candidate therapeutic agents for a subject. For example, a subject, such as a mouse, rat, dog, primate, or human, can be contacted with a candidate agent, its organ or biopsy can be prepared by the subject method, and the prepared specimen can be analyzed microscopically for one or more cellular or tissue parameters. A parameter is a quantifiable component of a cell or tissue, particularly one that can be accurately measured, preferably in a high-throughput system. A parameter can be any cellular component or cellular product, including a cell surface determinant, a receptor, a protein or its conformation or post-translational modification, a lipid, a carbohydrate, an organic or inorganic molecule, a nucleic acid, such as mRNA or DNA, or a moiety derived from such a cellular component, or a combination thereof. While most parameters provide a quantitative readout, in some cases, semi-quantitative or qualitative results are acceptable. The readout may include a single determined value or may include a mean, median, variance, or the like. Typically, a range of parameter readouts is obtained for each parameter from multiplexes of the same assay. Variability is expected, and the range of values ​​for each set of test parameters is obtained using standard statistical methods, with common statistical methods used to provide a single value. Thus, for example, one such method may include detecting cell viability, tissue angiogenesis, the presence of immune cell infiltration, effects that alter disease progression, etc. In some embodiments, the screen involves comparing the analyzed parameters with those from a control or reference sample, e.g., a similarly prepared sample from a subject not in contact with the candidate drug. Candidate drugs of interest for screening include known and unknown compounds encompassing numerous chemical classes, primarily organic molecules, which may include organometallic molecules, inorganic molecules, gene sequences, etc. Candidate drugs of interest for screening also include nucleic acids, such as nucleic acids encoding siRNA, shRNA, antisense molecules, or miRNA, or nucleic acids encoding polypeptides. An important aspect of the present invention is evaluating candidate drugs, including toxicity testing, etc.Evaluation of tissue samples using the subject methods can include, for example, genetic, transcriptional, genomic, proteomic, and / or metabolomic analyses.

[0121] The subject devices, methods, and systems can also be used to visualize the distribution of genetically encoded markers in whole tissues at subcellular resolution, such as chromosomal abnormalities (inversions, duplications, translocations, etc.), loss of genetic heterozygosity, the presence of genetic alleles indicative of a predisposition to disease or good health, likely responsiveness to therapy, ancestry, etc. Such detection can be used, for example, in the diagnosis and monitoring of diseases, personalized medicine, and paternity studies, as described above.

[0122] Databases of analytical information can be compiled. These databases can include results obtained from known cell types, references from analyses of cells treated under specific conditions, etc. A data matrix can be generated, with each point in the data matrix corresponding to a readout from a cell, and the data for each cell can include readouts from multiple labels. The readouts can be the mean, median, or variance, or other statistically or mathematically derived values ​​associated with the measurements. Output readouts can be further refined by directly comparing them with the corresponding reference readouts. The absolute values ​​obtained for each output under identical conditions represent the inherent variability of the biological system and also reflect the variability of individual cells and between individuals.

[0123] Examples of Non-Limiting Aspects of the Disclosure The above-described aspects (including embodiments) of the present subject matter may be beneficial alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing, certain non-limiting aspects of the present disclosure, numbered 1-47, are provided below. As will be apparent to one of skill in the art upon reading this disclosure, each individually numbered aspect may be used or combined with any of the preceding aspects or any of the aspects following the individually numbered aspect. This is in accordance with the principles of the aspects. It is intended to provide support for all such combinations, and is not limited to the combinations of aspects explicitly provided below.

[0124] 1. A method for in situ gene sequencing of a target nucleic acid in a cell in an intact tissue, the method comprising: (a) contacting a fixed and permeabilized intact tissue sample with at least one pair of oligonucleotides under conditions that permit specific hybridization; the pair of oligonucleotides comprises a first oligonucleotide and a second oligonucleotide; each of the first oligonucleotide and the second oligonucleotide comprises a first region of complementarity, a second region of complementarity, and a third region of complementarity, and the second oligonucleotide further comprises a barcode sequence; contacting a first oligonucleotide, wherein a first complementary region of the first oligonucleotide is complementary to a first portion of a target nucleic acid, a second complementary region of the first oligonucleotide is complementary to the first complementary region of the second oligonucleotide, a third complementary region of the first oligonucleotide is complementary to the third complementary region of the second oligonucleotide, and a second complementary region of the second oligonucleotide is complementary to a second portion of the target nucleic acid, and wherein the first complementary region of the first oligonucleotide is adjacent to the second complementary region of the second oligonucleotide; (b) adding a ligase to ligate the 5' and 3' ends of the second oligonucleotide to form a closed nucleic acid circle; (c) pre-incubating the tissue sample with DNA polymerase for a time sufficient to allow uniform diffusion of the DNA polymerase throughout the tissue sample prior to performing rolling circle amplification; (d) performing rolling circle amplification by contacting the tissue sample with deoxyribonucleotide triphosphates, wherein the ligated second oligonucleotide serves as a template and the first oligonucleotide serves as a primer for a DNA polymerase, so as to generate one or more amplicons; (e) embedding the tissue sample in a hydrogel before or after any one of steps (a) to (d); (f) crosslinking one or more amplicons to a hydrogel; (g) purifying the hydrogel to enhance its transparency; (h) contacting one or more hydrogel-embedded amplicons having barcode sequences with a lead primer and a fluorescently labeled probe, wherein the probe comprises an oligonucleotide comprising a first thiol group covalently attached to a fluorophore comprising a second thiol group by a disulfide bond between the first thiol group and the second thiol group; (i) ligating a lead primer and a fluorescently labeled probe, wherein ligation occurs only if both the lead primer and the fluorescently labeled probe are complementary to adjacent sequences of the same amplicon; (j) contacting the hydrogel with an anti-fade buffer containing an antioxidant; (k) imaging one or more hydrogel-embedded amplicons to determine the location of target nucleic acids in cells in the intact tissue undergoing in situ gene sequencing, wherein the imaging is performed in the presence of an anti-fade buffer; (l) contacting the hydrogel with a reducing agent to result in reduction of disulfide bonds and cleavage of the fluorophore from the probe; (m) removing the fluorophore from the hydrogel; (n) Repeat steps (h) to (m) A method comprising:

[0125] 2. The method of claim 0, wherein the antioxidant is N-propyl gallate. 3. The method of embodiment 1 or 2, wherein RNA endogenous to the tissue sample is first bound to the hydrogel prior to step (a). 4. The method of any one of aspects 1-3, wherein said purification of the hydrogel is carried out before or after any one of steps (a)-(d). 5. The method of any one of aspects 0 to 4, wherein the cell is present in a population of cells.

[0126] 6. The method of embodiment 5, wherein the population of cells comprises multiple cell types. 7. The method of any one of aspects 0-6, wherein the target nucleic acid is RNA or DNA. 8. The method of embodiment 7, wherein the RNA is mRNA. 9. The method of any one of aspects 0-8, wherein the second oligonucleotide comprises a padlock probe. 10. The method of any one of aspects 0-9, wherein the first complementary region of the first oligonucleotide has a length of 19-25 nucleotides.

[0127] 11. The method of any one of aspects 0-10, wherein the second complementary region of the first oligonucleotide has a length of 6 nucleotides. 12. The method of any one of aspects 0-0, wherein the third complementary region of the first oligonucleotide has a length of 6 nucleotides. 13. The method of any one of aspects 0-0, wherein the first complementary region of the second oligonucleotide has a length of 6 nucleotides. 14. The method of any one of aspects 0-0, wherein the second complementary region of the second oligonucleotide has a length of 19-25 nucleotides. 15. The method of any one of aspects 0-0, wherein the third complementary region of the second oligonucleotide has a length of 6 nucleotides.

[0128] 16. The method of any one of aspects 0-0, wherein the first complementary region of the second oligonucleotide comprises the 5' end of the second oligonucleotide. 17. The method of any one of aspects 0-0, wherein the third region of complementarity of the second oligonucleotide comprises the 3' end of the second oligonucleotide. 18. The method of any one of aspects 0-0, wherein the first region of complementarity of the second oligonucleotide is adjacent to the third region of complementarity of the second oligonucleotide. 19. The method of any one of aspects 1-0, wherein the barcode sequence of the second oligonucleotide provides barcoded information for identification of the target nucleic acid. 20. The method of any one of aspects 1-0, wherein contacting the fixed and permeabilized intact tissue comprises hybridizing a plurality of oligonucleotide primers having specificities for different target nucleic acids.

[0129] 21. The method of aspect 0, wherein the second oligonucleotide is provided as a closed nucleic acid circle, and the step of adding a ligase is omitted. 22. The method of any one of aspects 0-21, wherein the deoxynucleotide triphosphate comprises an amine-modified deoxynucleotide triphosphate. 23. The method of embodiment 22, wherein the amine-modified deoxynucleotide triphosphate comprises an acrylate N-hydroxysuccinimide moiety modification. 24. The method of any one of aspects 0-23, wherein embedding comprises copolymerizing one or more amplicons with acrylamide. 25. The method of any one of aspects 0-24, wherein the target nucleic acid is substantially retained in the hydrogel after purification.

[0130] 26. The method of any one of aspects 1-25, wherein purifying comprises substantially removing a plurality of cellular components from the hydrogel-embedded tissue sample. 27. The method of aspect 25 or 26, wherein the purifying comprises substantially removing lipids and proteins from the hydrogel-embedded tissue sample. 28. The method of any one of aspects 0-27, wherein imaging comprises imaging the one or more hydrogel-embedded amplicons using confocal microscopy, two-photon microscopy, light field microscopy, intact tissue expansion microscopy, and / or CLARITY™ Optimized Light Sheet Microscopy (COLM). 29. The method of any one of aspects 1-28, wherein the intact tissue has a thickness of 5-20 μm. 30. The method of any one of aspects 1-28, wherein the intact tissue has a thickness of 50-200 μm.

[0131] 31. The method of any one of aspects 1-30, further comprising sectioning the intact tissue and placing the section of intact tissue flat on a surface. 32. The method of embodiment 31, wherein a metal ring is pressed down onto the section and used to transfer the section flat onto the surface.

[0132] 33. A fluid system comprising: a) a device comprising a non-pressurized sample chamber; b) a lid, the lid covering the top of the sample chamber; c) an interface tube, a first end of the interface tube being held by the lid and a second end of the interface tube being positioned above the tissue sample in the sample chamber sufficiently close to the bottom edge of the sample chamber so that liquid in the sample chamber remains in contact with the bottom of the interface tube for as long as liquid remains in the sample chamber; d) a fluid line coupled to a first end of the interface tube such that fluid flows through the fluid line into the interface tube and out the second end of the interface tube onto the tissue sample in the sample chamber; e) a pump; f) an imaging system; g) a processor unit configured to perform the method according to any one of aspects 0 to 32; A fluid system comprising:

[0133] 34. The fluidic system according to embodiment 33, wherein the sample chamber is a well of a multi-well plate or slide chamber. 35. The fluidic system of aspect 33 or 34, wherein the interface tube is positioned at a polar angle relative to the tissue sample plane. 36. The fluidic system of any one of aspects 33-35, further comprising a cryostat that maintains the sample chamber at cryogenic temperatures. 37. The fluid system of any one of aspects 33-36, further comprising a multi-way selector valve interfaced with the pump. 38. The fluidic system of aspect 37, further comprising a reagent tray comprising one or more containers or wells containing reagents for performing in situ genetic sequencing of target nucleic acids in cells in intact tissue, the reagents being fluidly connected to the multi-way selector valve.

[0134] 39. An adjustable stage supporting a reagent tray, a cooler, and a position sensor for enabling movement of the adjustable stage to enable selection of a reagent from the reagent tray. 39. The fluid system of embodiment 38, comprising: 40. The fluidic system of any one of aspects 37-39, further comprising a buffer tray comprising one or more containers or wells containing a buffer, the buffer fluidly connected to the multi-way selector valve. 41. The fluidic system of any one of aspects 38-40, wherein the reagent tray and / or buffer tray is disposable. 42. The fluid system of any one of aspects 37-41, further comprising a waste container fluidly connected to the multi-way selector valve. 43. The fluid system of any one of aspects 33-42, wherein the pump is a syringe pump.

[0135] 44. A fluidic system according to any one of aspects 33 to 43, wherein the imaging system comprises a microscope that performs confocal microscopy, spinning disk confocal microscopy, light sheet microscopy, lattice light sheet microscopy, or light field microscopy. 45. The fluid system of any one of aspects 33-44, further comprising a container surrounding the fluid system, the container blocking ambient light. 46. ​​The fluid system of embodiment 45, wherein the container enclosing the fluid system comprises one or more doors or drawers. 47. The fluid system of aspect 45 or 46, wherein the container surrounding the fluid system is an acoustically attenuating container. 48. The fluidic system of any one of aspects 33-47, further comprising means for cryosectioning the tissue sample. [Example]

[0136] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0137] All publications and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0138] The present invention has been described with reference to specific embodiments discovered or proposed by the inventors to comprise preferred modes for carrying out the invention. Those skilled in the art will understand, in light of this disclosure, that numerous modifications and variations can be made in the specific embodiments exemplified without departing from the intended scope of the invention. For example, codon redundancy allows for changes in the underlying DNA sequence without affecting the protein sequence. Biological functional equivalence considerations allow for changes in protein structure without affecting biological activity in kind or amount. All such modifications are intended to be within the scope of the appended claims.

[0139] Example 1 New methods for intact tissue sequencing chemistry and processes Introduction STARmap is a sequencing process in which target nucleic acids are selectively labeled with barcodes, which are amplified by ligation prior to subsequent multiple rounds of readout. The initial publication of STARmap demonstrated the use of combinatorial sequencing on sample sizes as large as 0.0034 cubic millimeters and sequential sequencing on sample sizes as large as 0.238 cubic millimeters. Collecting these data required significant human labor, with the sequencing readout stage alone requiring several days of continuous labor for a single sample. An entire adult mouse brain is approximately 400 cubic millimeters, or over 110,000 times the volume of previously demonstrated combinatorial sequencing samples. As intact tissue sequencing approaches scale to increase capacity and throughput, maximizing their robustness (to prevent compound accumulation of errors) and enhancing parallelism and throughput are crucial. This is especially true as these techniques transition from the laboratory to clinical and industrial workflows.

[0140] The new method described herein targets several distinct robustness and throughput aspects of previous STARmap techniques, including both sequencing chemistry and automated execution of sequencing. We refer to this new method as "Clinical and Industrial-Scale Sequencing of Intact Tissues (CISITS)." The chemistry aspects of the new method focus on increasing the signal-to-noise ratio (SNR) of labeled molecules, eliminating carryover signal across rounds, achieving reliable efficiency and chemical success across protocols, and enabling combinatorial sequencing in thick tissues. The new automation method includes a combination of a fluidics system in which the sample chamber is no longer pressurized, a system integration method for high system robustness, and a method for parallelization across samples. The CISITS software control method manages the automated fluidics system and orchestrates a robust fluid exchange process while preserving the sequencing workflow in the event of failure. CISITS integration with microscope hardware simplifies and accelerates user interaction and provides system integration that enables large-scale field-of-view acquisition for increased throughput.

[0141] CISITS (1) A new method for high sequencing SNR In the original version of STARmap, amplicons are labeled during sequencing by attaching fluorescently labeled oligos to the appropriate amplicons so that they remain attached at room temperature after washing. Both post-ligation washing and imaging used a "wash and imaging" buffer formulated with 2x SSC, 10% formamide, and water. Imaging in this buffer suffered from photobleaching and loss of signal from the fluorophores with longer exposure times. This was problematic both in early rounds of sequencing (when signal is highest) due to image tiling with a wide field of view, which increases the total amount of light delivered to the edges of the field of view and reduces the SNR and detectability of individual amplicons. This is particularly problematic in later rounds of sequencing, which suffer from a characteristic decline in SNR due to the nature of SEDAL sequencing chemistry. In later rounds, particularly for long-wavelength fluorophores, a complete barcode read is required for successful sequencing, so poor SNR begins to impair the ability to extract high-quality reads from molecules, reducing the overall yield of sequenced molecules.

[0142] The washing and imaging buffer formulation was modified to include an antioxidant compound, such as N-propyl nitrate. For example, the new composition of the washing and imaging buffer was 2x SSC, water, and 10% N-propyl gallate dissolved in 20% formamide, resulting in a final concentration of 10% formamide and 2% N-propyl gallate. The inclusion of an antioxidant compound in the washing and imaging buffer acts as an anti-bleaching agent for the fluorophores. This reduces photobleaching during tiling and significantly enhances the SNR of sensitive fluorophores, allowing for higher SNR imaging of thicker samples. For a fixed exposure time with an antioxidant compound, the concentration of unbleached fluorophores per dot was increased over the exposure. The inclusion of an antioxidant also eliminates the reducing return effect of longer exposure times (caused by the limited phosphor lifetime before bleaching), allowing for a linear increase in SNR with longer exposure times as needed.

[0143] (2) Improved signal separation from round to round In the sequential readout approach to STARmap sequencing, barcoded amplicons from individual genes are probed in only a single round, ensuring that a given color channel strictly conveys information about genes of different interest in each round. This sequential readout approach is used in thicker tissue sections, where it is more difficult to control amplicon size and maintain amplicon separation across rounds, or in samples where fewer genes need to be read out and volumetric throughput is emphasized, which can be achieved by using lower-size objectives and camera binning, such as 4x4 binning of pixels per image. The success of this sequential approach requires that signals from a given color channel are completely eliminated from round to round. Otherwise, remaining signals from, for example, highly expressed genes in one round may be mistaken for signals from low-expressed genes in the next round.

[0144] This was resolved by increasing the formamide concentration from 60% to 80% and increasing the length and number of formamide stripping cycles for every round. However, this did not result in complete signal removal for all genes, although the efficiency was affected by room temperature variations and could add significant additional time per sample. The additional formamide exposure could potentially compromise sample quality over time and sequencing rounds.

[0145] To more completely eliminate round-to-round signal, another aspect of CISITS involves SEDAL-based sequencing readout combined with fluorophore cleavage (as opposed to probe stripping). The inclusion of a thiol bond between the fluorophore and the remainder of the oligo allows for fluorophore cleavage in a reducing environment, such as TCEP solution, which is used in other in situ sequencing approaches. However, in STARmap's SEDAL sequencing, high SNR labeling of amplicons occurs during the ligation reaction, and the ligation buffer contains the reducing agent DTT, which we found dramatically reduced signal from thiol-containing fluorescent oligos.

[0146] CISITS reformulates the SEDAL sequencing / ligation buffer to remove the DTT component. We found that thiol-containing fluorophore-labeled oligos can be conjugated to target amplicons with little difference in specificity or SNR compared to non-thiol-containing fluorescent oligos. Additionally, the removal of DTT allows thiol-containing fluorophore probes to remain stable in the ligation mixture for at least 24 hours at 4°C, with minimal loss of subsequent labeling efficacy, facilitating automation. After imaging a sequencing round, adding a buffer containing the reducing agent TCEP for 30 minutes completely eliminates punctate fluorescent signals, and a subsequent brief PBSTw wash removes any diffuse fluorescent signals.

[0147] This aspect of the new method has the advantages of robust signal removal, rapid round times with reduced signal removal / washing times, and particularly consistent tissue robustness, allowing for a higher total number of rounds without compromising sample integrity. The use of SEDAL sequencing with thiol-based chemical cleavage in CISITS combines the specificity and SNR advantages of SEDAL sequencing with the robustness and rapidity of round-to-round cycling from fluorophore cleavage.

[0148] (3) New methods for robustness in the CISITS protocol before and during sequencing Tissue section collection In combinatorial sequencing of intact tissues, fresh frozen tissue sections are collected by cryosectioning and placing the sections onto fresh, binding silane-coated wells or slides. To facilitate high-throughput, parallel processing of samples, it is preferable to collect samples in multiwell plates, such as 24-well plates. However, transferring frozen sections to the bottom of silane-coated plates without tissue disruption, folding, or tearing can be challenging, potentially interfering with subsequent data collection and analysis. Furthermore, tissue collection and preparation prior to sequencing readout must be highly parallelized across samples, making optimal sample collection essential. The STARmap method does not detail a robust sample collection process, and standard approaches result in a very high failure rate per section, lost sections, and inefficient use of plate space.

[0149] To ensure that frozen sections are placed flat on the surface used for downstream processing, as part of CISITS, a novel method was devised in which the sample collection well is kept cold in the cryostat along with a small, thin-walled metal ring, 8 or 10 mm in diameter. After the section is collected, the metal ring is pressed down onto the section so that the edge of the ring is in even contact with the cryosectioning medium surrounding the section. The ring and flat section can then be picked up using forceps and transferred to a suitable surface, which has been pre-warmed with a finger underneath. The metal ring facilitates precise flat placement of the section on the silane surface, and the warmth from the finger separates the section from the ring and adheres it to the well. This novel method significantly improves section integrity and throughput.

[0150] CISITS formulation in acrylic acid NHS ester buffer Previous formulations of AA NHS ester buffer in the STARmap method were not pH buffered. In the new CISITS method, which includes 20 mM MOPS pH 7.7 in the AA NHS ester buffer, the pH stabilizes the buffer at a more optimal pH for the AA NHS ester, making the subsequent NHS ester reaction reliable and efficient.

[0151] The CISITS protocol for reaction robustness It has been found that some aspects of sequencing chemistry require fresh reagents for robust high-throughput use over multiple samples and days of sequencing, which is particularly important for scalable, automated sequencing.

[0152] In the new CISITS method, polymerization II, RCA, and ligation mixtures must be made fresh for use in the CISITS process prior to sequencing.

[0153] Ligation mixtures for SEDAL sequencing rounds can be premixed and stored at 4° C. for at least 24 hours. Signal is optimal when SEDAL ligation round mixtures are made fresh every 24 hours of sequencing, provided they are stored at 4° C. During automated sequencing, additional SEDAL reagents for rounds >4 may be freshly added to the sequencing device after several rounds have been run.

[0154] TCEP buffer is best used when made fresh before each sequencing run, but maintains efficiency across sequencing cycles.

[0155] (4) A new method for combinatorial labeling of nucleic acids in thicker tissues The main limitation to the throughput of the STARmap method is the combined The main drawback of this method is the inability to perform sequencing on a single sample. This limitation stems from several factors. First, because an enzymatic reaction is required to ligate SNAIL probes and amplify the ligated probes using rolling circle amplification (RCA), the penetration of the enzyme into the sample and its subsequent efficiency can be severely limited. An additional limitation is the uniformity of the size control of the RCA amplicons. Due to the difficult diffusion characteristics of thicker tissues, it is not possible to obtain uniform amplicons of a suitable size for multiple rounds using the conventional STARmap method. Furthermore, the cost of obtaining probes suitably modified for direct incorporation into the hydrogel (prior to ligation and amplification) can be prohibitive for a large number of genes.

[0156] New methods have devised several approaches to overcome these limitations, enabling large-scale sequencing of tissues. These new methods offer several different means for combining thick sections and obtaining high-throughput sequencing.

[0157] In this first example of the new CITSIT method, probes containing appropriate terminal modifications, such as acrydite, are used for hybridization, as in the STARmap method. These may be purchased individually, modified in the laboratory, or obtained as a pool of oligos. After hybridization, a hydrogel is formed before digestion, ligation, and RCA. After digestion and ligation, amplification is performed using a new method to achieve sufficiently uniform ligation and amplification efficiency across the sample. First, an RCA mixture is formulated to contain enzymes and buffer but exclude dNTPs, thereby preventing any amplification. This mixture is added to the ligated sample and allowed to diffuse uniformly through the sample over the course of, for example, two days. Removing dNTPs allows the slow enzyme diffusion process to proceed to equilibrium. After the RCA mixture without dNTPs is added and allowed to equilibrate, an RCA mixture with an excess concentration of dNTPs is added to the sample. Because dNTPs are small molecules and have a high concentration gradient outside the sample, they rapidly equilibrate within the sample at room temperature. For example, after 30 minutes of equilibration with an RCA mixture containing dNTPs at room temperature, the temperature is raised to an optimal temperature for amplification, and amplification proceeds for several hours to produce uniform and appropriately sized amplicons for subsequent multiple rounds of combinatorial sequencing.

[0158] In the second example of the new method, endogenous RNA is first bound to a hydrogel prior to hybridization. This is achieved by covalently mediating interactions with endogenous RNA and polymerizing it into the hydrogel, either through conjugation chemistries such as label X, or by binding to the 5' methylated RNA mRNA cap, or by highly stable hybridization to the polyA tail, or by short condensed probes that can be individually displaced by subsequent target hybridization but collectively serve to retain the endogenous RNA in the hydrogel with sufficient long-term stability. After formation of the RNA-embedded hydrogel, the sample can be actively digested and permeabilized to yield a transparent hydrogel containing mostly endogenous RNA. This resulting hydrogel has superior diffusion properties compared to undigested tissue. Probe hybridization, ligation, and RCA can then proceed with moderate incubation times, while the method for homogeneous RCA amplification described in the first example ensures compatibility with downstream combinatorial sequencing. Following RCA, the amplicons are ligated to hydrogels following approaches previously described with the thin-section STARmap method for long-term stability before proceeding to combined multi-round sequencing.

[0159] Due to the orders of magnitude increase in thickness of these samples compared to thin section methods, incorporating N-propyl gallate or other antioxidant compounds in the washing and imaging buffers is essential to prevent photobleaching if large depths of the sample in Z are to be imaged.

[0160] A new automated fluid system hardware approach for CISITS. Previous automated methods for STARmap sequencing consisted of a fluidic system in which sequencing reagents were pushed and pulled across samples in a pressurized, air-free flow cell. Accurate and robust functioning of this flow cell-based system required removing air bubbles from the fluid lines, properly managing the flow of reagents in the flow cell so that reagents were fully exchanged at each step of the sequencing run, properly priming the system so that the correct amount of each reagent was loaded, and properly sealing the flow cell when connecting the fluids. Failure to meet any of these requirements at any point during multiday sequencing can lead to sequencing failure. Meeting these requirements is complicated by the heterogeneous shapes and sizes of samples on the flow cell. Additionally, a fundamental incompatibility exists between the STARmap method for pre-sequencing sample preparation and the previously described methods for automated STARmap sequencing in a flow cell. That is, while existing STARmap methods allow for the parallel preparation of many samples attached to individual wells of a multiwell plate, they do not specify any method for transferring the hybridized, ligated, amplified, and gelled samples to the flow cell, which is difficult due to the adhesive nature of the samples to the bottom of the sample preparation plate. No method has been described for implementing the STARmap sample preparation method on a flow cell from scratch, which suffers from evaporation and liquid management issues in addition to the loss of ease of parallelization across individual samples. Finally, previous STARmap automated methods described sequencing only a single sample (or flow cell) at a time, severely limiting the throughput of the method. We have developed a new method to address many of these robustness and throughput issues of previous methods.

[0161] CISITS Fluid System A new method for sample interfacing The CISITS fluidic system adds and removes sequencing reagents from the sample, but unlike previous methods, this new method uses a non-pressurized sample chamber that can accommodate air above the sample. Relaxing the requirement for reagent flow relative to the sample in favor of simply immersing the sample in the reagents greatly improves the robustness and simplicity of the system, while also greatly facilitating parallelization of automated sequencing across different sample chambers.

[0162] In this system, one or more samples are located in a sample chamber, which can be a glass-bottom well of a multiwell plate (for optimal compatibility with the CISITS method before sequencing) or any glass-bottom chamber that allows the air in the chamber to exchange with room air and relieve flow pressure. In one example, samples are prepared using the CISITS method in the wells of a 6-, 12-, or 24-well multiwell glass-bottom plate. The fluidic system is connected to the sample via a custom well-plate lid, which holds a rigid fluidic tube (such as a metal tube) immediately above the sample near the bottom edge of the sample well (the interface tube). A fluidic line connects to this tubing so that fluid flows through the fluidic line, into the interface tube, and onto the sample in the well. To remove fluid from the sample, the fluid is aspirated from the sample well through the interface tube. Different custom lids for the CISITS fluidic system are designed to accommodate different sample chamber formats, including multiple arrangements of multiwell plates or slide chambers in various different formats. In the case of multi-well plates, the system may simultaneously couple to multiple different sample wells, providing access to different sample wells (which are sequenced sequentially), or alternatively providing parallel sequencing capabilities across wells (see below).

[0163] The custom CISITS lid, which connects the fluidic system to the sample, can be easily placed on or removed at any time to allow air exchange between the sample chamber or plate. The fluid interface tube is not sealed onto the sample well. However, the fluid interface tube is precisely positioned within the lid so that the liquid in the sample chamber remains in contact with the bottom of the tube (through cohesion and adhesion) as long as the liquid remains in the chamber. This ensures that all or nearly all of the reagent is removed from the sample well with each reagent removal step, resulting in a robust automated process. Sufficient space is left between the bottom of the sample interface tube and the sample chamber so that liquid added to the sample does not pressurize the interface tube but instead spreads throughout, immersing the sample. The sample interface tube can be positioned at a polar angle to the plane of the sample so that many tubes can fit into a container with many different sample chambers, each with its own interface tube. Alternatively, custom CISITS lids can allow users to place single or multiple interface tubes into several desired sample chambers, ensuring that the tubes are guided into the precise position that results in robust fluid exchange. These custom lids can be machined or 3D printed to specifications and can be consumable or reusable.

[0164] A new method for continuous fluid flow through reagent selection, sample, and waste The fluidic system for CISITS centers around a syringe pump, which draws desired volumes of reagents into the pump through a series of selector valves. For single-sample operation, the pump in this new method has several modes of operation. It can draw reagents from a multiport selector valve coupled to a reagent reservoir, or it can draw reagents from a sample well through interface tubing. Once the pump draws in a volume, it can either push the volume through the interface tubing to the sample well, push the volume to a waste container, or push the volume back into a reagent reservoir (which may be used for cleaning purposes). These different modes of operation are mediated by a multiway selector valve (e.g., three-way) interfaced with the pump, which switches which fluid line is currently open. Thus, an example of a typical mode of operation for continuous sequencing would be to open only the pump selector to the reagent selector line, open the desired multiport selector line for the desired reagent, draw the selected reagent from the reagent reservoir through the multiport selector and into the pump, allowing equilibration to occur to control for varying flow times due to different liquid properties, open the pump selector to only the sample well, raise the syringe pump to push the reagent through the sample fluid line and interface tubing to bathe the sample, draw a small amount of air into the pump in place of the reagent and push it into the sample, flush the sample fluid line of any remaining reagent, repeat this process to fully deliver the desired amount of reagent to the sample, wait for any incubation time as part of the CISITS sequencing process, lower the pump, suck liquid from the sample back into the pump through the interface tubing to allow equilibration, open only the pump selector port to waste, raise the pump to push the contents to waste, and repeat this liquid removal process for a sufficient time to fully remove the desired amount from the sample. This can be considered a reagent addition, incubation, and removal cycle in the typical operation of the CISITS method. The cycle is repeated with either the same reagent or a different reagent as the sample progresses through the CISITS sequencing process.This cycling process in the fluidic system differs from the flow cell process in that the sample is left without liquid for a short period of time. However, because the CISITS method uses tissue embedded in a hydrogel, the sample does not dry out over this timescale and does not need to be constantly immersed. Furthermore, because the sample is attached to the bottom of the chamber and imaged with an inverted microscope, air cannot travel between the bottom of the well and the sample, so air bubbles are not an issue with this method.

[0165] This non-pressurized sample chamber method for fluidic systems allows for various robust methods, such as the aforementioned movement of air through the system after reagent addition, to ensure complete addition of reagent to the sample. Additionally, it allows for the addition of a larger volume than can be drawn by the pump. This allows for reagent, providing additional air volume to the pump, but ensuring that any limited amount of reagent is completely drawn from the reagent container. This method of fluidics can bypass the sample by drawing liquid into the pump and pushing it directly into a waste container, facilitating the cleaning process; it can draw water, diluted bleach, or other cleaning fluid into the pump, clean it, and push it directly out to waste. Alternatively, water or other cleaning fluid can be pushed into the reagent container as needed, for example, to clean the reagent fluid lines.

[0166] A new method for parallel fluid flow. This new CISITS method, in which the sample chambers are not pressurized, greatly facilitates parallel fluid delivery across multiple different sample chambers. In this new method, additional selector valves mediate the fluid lines from the pump to the samples. These selector valves can be opened singly (allowing addressable access to different sample wells) or multiple selector valves can be opened simultaneously, allowing fluid to be delivered to multiple samples in parallel by the pump. By connecting the output fluid lines from this sample selector valve to each different sample interface tube and sample, parallel sequencing using CISITS can be achieved, providing substantial throughput in sequencing multiple samples. For example, instead of a serial sequencing process of sequencing one sample at a time, each taking approximately 48 hours, many samples can be sequenced simultaneously or alternately, allowing the image acquisition system to become the limiting process instead of the sequencing cycle or incubation time. For example, for a single sequencing round (out of seven or more), reagent addition, incubation, and removal can collectively consume 80% of the round completion time, while imaging consumes 20%. Operating in parallel across more than five samples yields a 4x throughput speedup (it takes 1x as long to image all five samples in each round), and alternatively, operating reciprocally or alternatingly yields a nearly 5x speedup (less than one round of alternation time) since image acquisition is almost always performed following the first round. Thus, this new method yields a nearly linear increase in throughput for intact tissue sequencing with the number of samples sequenced in parallel up to the number of samples where the image acquisition system becomes limited.

[0167] A new approach for reagent management systems. In previous STARmap automated sequencing methods, reagents were kept cooled through different sample tubes in a cooling block or in tubes at room temperature. Each reagent was directly coupled to the fluidic system, requiring the user to switch between reagents between runs by directly coupling and uncoupling them from the fluidic system.

[0168] In the new method, the reagent management system abstracts this process for the user, simply providing a disposable reagent tray that can be easily placed into the fluidic device. The device places the appropriate portion of this reagent tray on the internal cooling block and automatically couples the fluid lines to the reagents. The reagent containers or wells in the reagent tray have sufficient heat transfer properties to allow coupling with the chiller unit. After the CISITS run / automated fluidic actuation is complete, the tray can be discarded and a new tray can be used for a new run. The user simply fills the labeled areas of the reagent tray with the appropriate reagents before placing it into the fluidic system. The reagent tray can vary in volume for different reagents depending on the system throughput and whether samples are being sequenced by the CISITS method in parallel or serially.

[0169] To ensure freshness of the sequencing reagents, the tray may optionally be partially removed during a sequencing run to deposit fresh mixed SEDAL reagents for the next few rounds.

[0170] This new method for reagent management simplifies and speeds up the initialization of a CISITS run This provides a robust process that prevents users from disrupting the normal operation of the system fluids.

[0171] A new method for fencing the CISITS sequencing system The entire sequencing system is enclosed by a light-tight enclosure that provides appropriate doors and drawers for accessing the various internal components of the system. Some drawers are intended for normal use of the system, such as drawers for loading and removing samples from the system and drawers for loading and removing reagents from the system. Other components of the enclosure provide easier access for service professionals to modify, upgrade, or repair internal parts of the system. The enclosure may include sound attenuation.

[0172] New methods for robust operation of fluid systems The CISITS automated sequencing system includes several methods to ensure robust automated operation and a low failure rate. These methods include, for example, a fluid flow detector through the sample fluid line to ensure fluid delivery, whose activity can halt sequencing to prevent catastrophic damage; an absorbent material surrounding the imaging objective and electrodes to detect reagent spillage from the sample container; confirmation that the cooler has reached the appropriate temperature before loading reagents; a check to detect proper loading of the CISITS reagent tray into the system; periodic system cleaning performed automatically at the end of each sequencing run to prevent clogging of the fluid lines due to salt or other material buildup; and intermittent cleaning performed before runs if the system has not been used recently. The automatic cleaning performed at the end of a sequencing run involves pumping a dilute bleach or other cleaning solution into the system, which is then discarded, followed by multiple rounds of water washing to completely remove the bleach or cleaning solution. After the automated procedure is complete, the system prompts the user to place a wash sample container matching the format previously used by the user so that bleach or wash solution and water can be passed through the sample fluid lines and tubing to wash the sequenced samples without damaging them, or this wash can be performed automatically on the sample container with the user's consent. Finally, air is passed through the system to remove any remaining liquid in the fluid lines. Intermediate washes can be initiated by the user using a wash sample plate between sequencing runs, provided that a wash reagent tray with wash buffer and / or diluted bleach and water is placed in the device. These wash cycles push wash solution through the pump, both the sample fluid lines, and the reagent fluid lines, remove and discard the wash solution, perform multiple wash cycles with water, and then flush the system with air.

[0173] A new method for system integration of fluid dynamics and microscopy enabling CISITS The new CISITS method requires an image acquisition system or microscope capable of rapidly acquiring images of the sample at submicron resolution across the sample depth for proper dot separation during combinatorial sequencing. Several different microscopes and microscope styles can be compatible with the CISITS fluidic system, including confocal microscopy, spinning disk confocal microscopy, light sheet microscopy, lattice light sheet microscopy, and light field microscopy, depending on whether combinatorial sequencing with CISITS (greater requirements for resolution as individual amplicons are resolved) or sequential sequencing with CISITS (fewer requirements for resolution) is being performed.

[0174] In one example, a spinning disk confocal microscope is coupled with an automated CISITS fluidics system, which communicates with the microscope system via software to synchronize or alternate image collection with the fluidics process. This communication can be continuous via a REST API, via TTL triggers, or by other software methods.

[0175] In one example, the microscope is a consumer product, communicating through software with the CISITS sequencing management and mechanically coupled to the automated fluidics system by the user simply placing a sample container on the microscope stage. Using the microscope image acquisition interface, the user controls imaging parameters and acquisition details, which are then triggered by automated sequencing at the appropriate time.

[0176] In one example, a microscope is a consumer product and is enclosed with a fluidic system such that the resulting enclosure functions as a single integrated system with an interface specified by the CISITS system.

[0177] In one example, a microscope is an OEM format, allowing microscope components to be more directly integrated with the automated fluidics system, allowing the entire system to be shipped in an integrated form and eliminating unnecessary mechanical parts that are typically part of a consumer product. The OEM microscope format allows for a more compact enclosure of the automated system. Direct integration facilitates the use of dedicated hardware for CISITS automation that directly interfaces with the microscope's imaging components. These include, for example, reagent leak repair and detection at the objective or objective turret. In particular, new methods for large-area or multi-area acquisition for CISITS, such as automated management of the liquid immersion interface between the objective glass and the sample chamber glass, are being explored. Automated management of the immersion interface, e.g., water, allows for large stage travel distances without losing immersion and proper refractive index matching, providing the ability to collect entire sections, and, importantly, allows the objective head to translate to multiple different wells without losing the water (or other) immersion interface.

[0178] New software and new methods for controlling the automated sequencing process Previous methods for automated STARmap sequencing described firmware code that controls individual fluidic components and suggested that this firmware code be coordinated by subsequent custom software on a controller computer.

[0179] New CISITS methods extend this process to a multilevel, hierarchical model of abstraction through system component control software compatible for parallel operation; integrate sequencing protocols into a single set of modular commands that accepts parameter files specifying relevant sequencing details (from combinatorial to sequential sequencing and variations of those protocols); provide a user interface for specifying, starting, pausing, and stopping sequencing runs and initiating wash runs; provide a network-accessible system dashboard that displays sequencing progress and metrics, including quality checks; and provide a method for a data management system to accommodate large datasets where space constraints prevent sequencing from progressing. These new methods provide a means for rapid, automated, and continuous acquisition of CISITS data.

[0180] Software methods for hierarchical abstraction and system control. The lowest level of firmware code is the individual controller for each device in the fluid system, e.g., a cooler block, pump, or selector valve. The Arduino or microcontroller contains firmware code that specifies the general operation of the individual hardware components and provides a series of macros that operate these hardware components according to input parameters. This microcontroller code is interfaced by a serial connection to a computer capable of image acquisition. The CISITS software running on the computer is itself hierarchical, providing multiple abstractions for the control of the various hardware components. In one example, this software is implemented in Python. It is implemented in the thon programming language. The low-level module provides an object-oriented model for programmatic interfacing with a hardware microcontroller / Arduino. Initialization of this object ("Robo") opens a serial connection with the microcontroller, provides a handle for subsequent communication via the serial interface connection, and resets the microcontroller device. Additionally, initialization sets basic parameters for device operation that are invariant across different sequencing protocols, such as maximum pump movement speed, maximum volume, and various time constants for fluid equilibration. The method providing the lowest level of abstraction in this object is the send command method, which communicates properly formatted serial command strings to the microcontroller and triggers the appropriate control macros. Higher-level methods in the object coordinate commands to the microcontroller that perform individual operations of the fluidic system, including getting and setting the cooler temperature, waiting until the cooler reaches the target temperature, moving the pump contents to waste, filling a specified volume of input reagent into a specified sample, draining a specified volume of a specified sample well to waste, and transferring reagents between reagent containers. The final key method of the object is closing the serial connection to the microcontroller. By establishing and maintaining this continuous connection with the RoboObject, the software blocks other programmatic threads' access to the microcontroller, ensuring that only a single thread can coordinate the fluid hardware at a time.

[0181] This control object is initialized and its methods are called by the sequencing module, which provides a higher-level framework for the specification of sequencing protocols. In one example of a CISITS software method, sequencing commands are organized in an "execution-blocking" paradigm, where individual operations are executed serially and the sequencing procedure waits for the program to complete each command. This provides a convenient and robust way to prevent conflicting commands from acting on the system simultaneously, particularly during incubation periods, which can block program execution for the allotted time. In this paradigm, progression through the CISITS protocol, including reagent addition and removal and incubation periods, is performed strictly sequentially following the execution of a script-like program. Multiple washes or additions of reagents occur within the program for every loop, and these are summarized in loops within the program across sequencing rounds.

[0182] In another example of the CISITS software method, parallel sample sequencing control is implemented as a multithreaded application, with individual processing threads acquiring locks on the sequencing hardware and asynchronously executing the required sequencing steps, one thread per sample well. In this mode of operation, imaging and fluid movement can occur simultaneously. In another example, threads operate asynchronously but communicate to optimize the timing of sequencing operations and meet strict incubation time constraints in the chemistry.

[0183] In another example of the CISITS software method, sequencing is performed in a state-dependent event loop paradigm, where the sequencing module describes progression through a series of states, some of which may occur in parallel, and the event loop executes this progression according to the state of the system, the program's position in the state flow, and the availability status of system components. This paradigm is particularly suited to the reciprocal or parallel operation of a sequencing device across multiple samples, where not all samples are absolutely synchronized in their sequencing phase. For example, a sample incubation command may be executed such that a timer is set and checked for a given sample; once the timer is complete, the program may continue with that sample. However, in the meantime, the state of a pump or valve may be available and commanded to operate on other samples while the first sample is incubating.

[0184] The sequencing module is responsible for the proper movement of fluids or accidental spills, etc. The sequencing loop or event loop also includes a check for an interface file that communicates to the sequencing thread whether sequencing should be paused or stopped. In the case of a pause, the sequencing module checks for the continued existence of the pause file every few seconds and proceeds with sequencing if it is no longer found. In the case of a stop interface file, the sequencing module reports to the log that a stop has been detected, resets the device, and terminates. Generally, when the sequencing device begins operation, it writes a file indicating that it is running, and deletes the file once it has completed operation or if it exists.

[0185] Finally, the sequencing module and its commands for running CISITS are wrapped by a high-level graphical user interface. If the sequencing device is not already in use, the user interface can run the sequencing (or cleaning) module as an independent program thread. Additional threads, including a sequencing progress dashboard thread and a data management thread, can be automatically started by this interface if they are not already running.

[0186] A software method for specifying parameters of CISITS sequencer operation. The sequencing module of the CISITS control software is parameterized with settings specific to a given sequencing protocol, such as combinatorial or sequential sequencing. The method is designed so that specification files for different sequencing methods fully parameterize the sequencer module's commands. While the default settings provide sufficient robustness for automated execution of the sequencer, the user interface offers an advanced mode in which these settings can be modified and saved. This model, which separates parameters from the primary sequencing method, allows for parameter recording, reproducibility, and reuse, simplifying the software to a common sequencing module.

[0187] Software methods for graphical user interface control of sequencing The graphical user interface (GUI) is the primary interface between the user and the CISITS sequencing device. It displays the status of the sequencing device during runtime and provides buttons to pause or stop the operation of the sequencing device. While running on a separate thread, the sequencing module repeatedly checks a file in its directory to indicate whether sequencing should be paused or stopped entirely, and allows communication between the user interface thread and the sequencing thread, which are otherwise independent, once sequencing is initiated. If sequencing or cleaning is not in progress, the GUI provides buttons to start a new sequencing run and a new cleaning run. It also indicates whether a cleaning needs to be performed based on the time elapsed since the last CISITS sequencing run. When a cleaning is initiated (the cleaning button is pressed), the GUI displays a message to the user to position the cleaning reagent tray and wash the sample container into the sequencing device. The user presses a button to confirm these are in place, and the cleaning cycle proceeds, notifying the user on the GUI of any errors. When sequencing is initiated (the Sequencing button is pressed), the user is provided with a text entry box for the experiment name, a drop-down list for selecting either combinatorial or sequential sequencing, and a drop-down list indicating the number of rounds to perform. The user can also click a button to display advanced options, including text entry boxes for default sequencing parameters for combinatorial or sequential sequencing, load parameters from a saved parameter file, or enter different values ​​and a filename to save a new set of custom sequencing parameters. The user can set the directory or location to transfer data to, including the option to set up automatic transfer to cloud storage. After entering the relevant information into the GUI, the user presses the Continue button to begin sequencing or the Cancel button to return to the home screen. Pressing the Start button causes the GUI to briefly open an interface object to the fluidics system, checking whether the cooler is already sufficiently cold. If the cooler is not yet cold, it begins cooling before disconnecting from the fluidics system. The GUI then guides the user through the remaining sequencing setup steps, including setting imaging parameters and loading reagents into the machine. Finally, the user can press the Start button to begin the sequencing operation, and the sequencing module is invoked as a thread with the entered or selected parameters. At that point, the GUI ensures that a local server thread is running to provide a network-accessible sequencer status dashboard and that a data transfer thread is running. After completing the main sequencing operation and pre-cleaning, the GUI prompts the user to replace the sample container with a cleaned sample container. Clicking the Continue button performs a final cleaning cycle of the sequencer.

[0188] A new software approach to system status dashboards Initializing sequencing with the CISITS software also launches a web server thread that serves as a dashboard for the sequencing device computer, accessible via the local network. This dashboard displays the status of the sequencing device, including any errors and the progress of the sequencing device on a given sample or on several samples. This includes which round of CISITS is being performed, the elapsed time, the estimated time to completion, data on round-to-round completion times, data on image acquisition times, and quality control metrics. This includes, for example, combinatorial sequencing, the distribution of dots detected in the first round per field of view per area, median dot size, dot distribution across color channels, and registration distances between rounds, depending on the sequencing, e.g., signal correlation across channels within a round, or signal correlation between rounds within a color channel. To prevent unauthorized network access to this dashboard, the dashboard requires a user to log in to view it, and the server limits the number of network requests that can be received from IP address blocking to reduce denial-of-service attacks on the acquisition computer. The dashboard does not provide any control over the sequencing device. Data from the dashboard is provided by the sequencer module, which records operations and times, for example via log files and files saved via an external processing pipeline, records computer quality control data on the sequencing data as it becomes available, and provides the dashboard with network locations where the quality control data can be obtained.

[0189] A new software method for acquiring and managing CISITS data. Large-area CISITS sequencing rounds, multiple sequencing rounds, and multi-sample or multi-chamber parallel sequencing all produce data per sequencing round per sample, which can fill a large portion of a large hard drive. For example, a single tile microscope acquisition of a region of interest in a CISITS sample can range from hundreds of gigabytes to several terabytes. Therefore, software methods are essential to actively move data from the acquisition hard drive onto a very large local storage drive pool or onto a large buffer drive, followed by a large network storage drive or cloud storage for subsequent downstream processing. These software methods can run as independent threads that check for available data and move it from the acquisition drive to its storage destination. These methods are essential for large-scale, high-throughput sequencing, because failure to properly manage data can quickly result in space being lost on the acquisition drive, stalling sequencing.

[0190] New software methods for integration with microscope platforms The CISITS software method integrates fluidic system operation with the operation of the microscope platform. In some examples, the microscope platform provides a separate image acquisition GUI, which can be used to set desired imaging parameters, field of view, tile, and position, triggered or initiated by continuous, TTL, REST, or other API commands from the sequencing software module. In this case, the sequencing module automatically communicates with the image acquisition software to initiate a user-specified image protocol at the correct time within the sequencing chemistry protocol and waits for that sample's acquisition to complete before starting a subsequent image acquisition or procedure. Alternatively, as with parallel sample well operation, the software can initiate image acquisition and proceed with other fluidic operations on other samples, periodically querying the image acquisition software for the completion status of the ongoing acquisition before starting a new queued acquisition. In other examples, particularly when the microscope platform is more directly integrated with the fluidic system, the CISITS software itself integrates sufficient microscope functionality within the GUI for the user to set desired image acquisition parameters before sequencing begins. In this case, the microscope functionality is implemented directly by the CISITS software, either by wrapping an external microscope control and interface library, such as MicroManager, or by directly integrating with the microscope system's acquisition software. Minimal CISITS software integration with the microscope platform includes setting pre-corrected acquisition filenames, running saved imaging protocols on the microscope system, obtaining acquisition completion status, operating the sequencer in parallel across multiple sample wells, and acquiring and setting stage positions. The CISITS software, which controls sequencing across multiple sample regions, allows users to specify the starting position of each different region to be imaged and link those regions to parallel well sequencing or multiple positions imaged sequentially within a single chamber.This is achieved during imaging setup where the software queries the microscope system for stage positions and saves the stage positions as a list of acquisition start positions for a given sample, or an external list of acquisition start positions where the sample chambers are in different parallel orders.

[0191] Purpose Described herein is the in situ nucleic acid sequencing technique from laboratory scale to clinical scale and industrial scale.The sample type of these techniques can include cultured cell layers, cultured organoids or the slices of cultured organoids, as well as animal biopsy samples or post-mortem animal tissue.It can be used to record the spatial location of target genes in these samples, the genetic "identity" or "type" of cells based on the pattern of expressed genes or the presence of marker genes, the location of exogenous genes such as anatomical or functional markers in cells or tissues, the change in RNA expression under experimental conditions, and can identify the spatial expression of genes in cells that express other orthogonal markers, including being used in genetic screening or engineering screening.

[0192] Advantages and improvements over existing methods, devices, or materials Compared to the previously described STARmap method, CISITS combines chemical improvements for SNR and process robustness, as well as automation (hardware and software) improvements for throughput and robustness, allowing for improved robustness and high-throughput sequencing of intact tissues.

[0193] STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was funded by Defense Advanced Research Projects This work was produced with government support under contract W911NF-14-2-0013 awarded by the Agency and contracts DA042012 and MH075957 awarded by the National Institutes of Health. The U.S. Government The present invention has certain rights.

Claims

1. 1. A method for in situ gene sequencing of nucleic acids in cells in a tissue sample, comprising: (a) contacting the tissue sample with a plurality of oligonucleotides under conditions that allow for specific hybridization; (b) pre-incubating the tissue sample with a polymerase in the absence of nucleotides; (c) performing an amplification reaction by contacting the tissue sample with a plurality of nucleotides to generate one or more amplicons from the plurality of oligonucleotides; (d) imaging the one or more amplicons to identify the nucleic acid; and A method comprising:

2. the plurality of oligonucleotides comprises a first oligonucleotide and a second oligonucleotide; (i) the first oligonucleotide comprises a first region that binds to a first portion of the nucleic acid; (ii) the first oligonucleotide binds to the second oligonucleotide, or (iii) the second oligonucleotide binds to the nucleic acid; or (iv) any combination of these is present; The method of claim 1.

3. The method of claim 2 , wherein the second oligonucleotide comprises a second region that binds to a second portion of the nucleic acid.

4. 4. The method of claim 3, wherein the first region is adjacent to the second region when the first oligonucleotide and the second oligonucleotide are bound to the first portion and the second portion of the nucleic acid, respectively.

5. The method of any one of claims 2 to 4, wherein the first oligonucleotide comprises a third region and the second oligonucleotide comprises a fourth region, and the third region is complementary to the fourth region.

6. 5. The method of claim 2, wherein the first oligonucleotide comprises a fifth region and the second oligonucleotide comprises a sixth region, and the fifth region is complementary to the sixth region.

7. 5. The method of claim 2, further comprising, after step (a), adding a ligase to the tissue sample to generate a closed nucleic acid circle with the second oligonucleotide.

8. The method of any one of claims 1 to 4, further comprising, after step (c), clarification of the tissue sample of a plurality of cellular components.

9. The method of claim 8 , wherein the cellular components include lipids, proteins, or a combination thereof.

10. The method of any one of claims 1 to 4, wherein the amplification reaction comprises a rolling circle amplification reaction.

11. The method of any one of claims 1 to 4, further comprising embedding the tissue sample in a hydrogel before or after any one of steps (a) to (c).

12. 12. The method of claim 11, further comprising crosslinking the one or more amplicons to the hydrogel.

13. The method of any one of claims 1 to 4, wherein the one or more amplicons comprise a barcode.

14. 14. The method of claim 13, wherein step (d) comprises detecting the barcode to identify the nucleic acid.

15. The method of any one of claims 1 to 4, further comprising adding a buffer containing an antioxidant to the tissue sample after step (c).

16. 16. The method of claim 15, wherein the antioxidant comprises N-propyl gallate.

17. The method of any one of claims 1 to 4, further comprising heating the plurality of oligonucleotides prior to step (a).

18. The method according to any one of claims 1 to 4, wherein the tissue sample is a formalin-fixed, paraffin-embedded tissue sample.

19. The method of any one of claims 1 to 4, wherein the tissue sample is a fresh frozen tissue sample.

20. The method according to any one of claims 1 to 4, wherein the nucleic acid is a ribonucleic acid.

21. 21. The method of claim 20, wherein the ribonucleic acid comprises messenger ribonucleic acid.

22. The method according to any one of claims 1 to 4, wherein the nucleic acid is a deoxyribonucleic acid.

23. The method of any one of claims 1 to 4, wherein step (d) comprises imaging the one or more amplicons using an imaging system.

24. The method of claim 23 , wherein the imaging system comprises a light sheet microscope.

25. 24. The method of claim 23, wherein the imaging system comprises a confocal microscope.

26. The method according to any one of claims 1 to 4, wherein the tissue sample has a thickness of 50 to 200 μm.

27. The method of any one of claims 1 to 4, further comprising adding a permeabilization reagent to the tissue sample prior to step (a).

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