Sequencing of intact tissues at clinical and industrial scales

Abstract

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 they modulate gene expression of nucleic acids in cells in intact tissue.

Description

Background Art

[0001] Biological samples contain complex and heterogeneous genetic information ranging from the scale of the length of individual cells to the scale of the length of the entire tissue. The spatial pattern of nucleic acids within cells can potentially reveal the characteristics and abnormalities of cell function, the cumulative distribution of RNA expression can potentially define cell type or function, and the systematic variation in the position of cell types within a tissue can potentially define tissue function. The combination of anatomical connectivity information encoded in nucleic acids and the cell type distribution across the entire tissue can span many sections of the tissue.

[0002] Therefore, techniques for in situ nucleic acid sequencing must be able to bridge the resolution as small as an individual molecule and the resolution as large as the entire brain. To efficiently collect and record this information across orders of magnitude of length differences, new inventions are needed to enhance the robustness, speed, 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 within intact tissue. Methods are also provided herein for screening candidate agents to determine whether the candidate agent modulates gene expression of nucleic acids in cells within intact tissue.

[0004] In one aspect, a method for in situ gene sequencing of a target nucleic acid in cells in intact tissue is provided, the method comprising: (a) contacting an immobilized 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, the third complementary region of the first oligonucleotide being complementary to the third complementary region of the second oligonucleotide, the second complementary region of the second oligonucleotide being complementary to a second portion of the target nucleic acid, and 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 ring; (c) pre-incubating the tissue sample with a 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 deoxyribonucleoside triphosphates such that one or more amplicons are generated, wherein the ligated second oligonucleotide functions as a template and the first oligonucleotide functions as a primer for the DNA polymerase; (e) embedding the tissue sample in a hydrogel either before or after any one of steps (a) to (d); (f) cross-linking one or more amplicons to the hydrogel; (g) purifying the hydrogel to enhance the transparency of the hydrogel; (h) one or more hydrogel-embedded amplicons having a barcode sequence,contacting with a primer and a fluorescently labeled probe, wherein the probe is a first thiol group covalently bonded to a fluorophore containing a second thiol group by a disulfide bond between the first thiol group and the second thiol group, contacting, comprising an oligonucleotide containing a thiol group; (i) ligating the primer and the fluorescently labeled probe, wherein ligation occurs only when both the primer and the fluorescently labeled probe are complementary to adjacent sequences of the same amplicon; (j) contacting the hydrogel with an anti-fading buffer containing an antioxidant; (k) imaging one or more hydrogel-embedded amplicons to determine the positioning of a target nucleic acid in cells in intact tissue undergoing in situ gene sequencing, wherein imaging is performed in the presence of the anti-fading buffer; (l) contacting the hydrogel with a reducing agent to effect reduction of the disulfide bond and cleavage of the fluorophore from the probe; (m) removing the fluorophore from the hydrogel; and (n) repeating steps (h) to (m).

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

[0006] In another aspect, a fluid system for automating the methods described herein to enable continuous operation is provided. In some embodiments, the system comprises a fluid device and a processor configured to perform the methods described herein.

[0007] In certain embodiments, there is provided a fluid system comprising: a) a device comprising an unpressurized sample chamber; b) a lid that covers the top of the sample chamber; c) an interface tube, wherein a first end of the interface tube is held by the lid and a second end of the interface tube is positioned over a tissue sample in the sample chamber in sufficient proximity to the bottom edge of the sample chamber such that, as long as liquid remains in the sample chamber, the liquid in the sample chamber remains in contact with the bottom of the interface tube; 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 onto the tissue sample in the sample chamber from the second end of the interface tube; 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 multi-well plate or a slide chamber. In some embodiments, the interface tube is disposed at a polar angle with respect to the tissue sample plane. In some embodiments, the fluid system further comprises a cryostat for maintaining the sample chamber at cryogenic temperatures. In some embodiments, the fluid system further comprises a multi-directional selector valve interfaced with the pump. In some embodiments, the fluid 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-directional selector valve. In some embodiments, the fluid 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-directional selector valve. In some embodiments, the reagent tray and / or the buffer tray are disposable. In some embodiments, the fluid system further comprises an adjustable stage for supporting the reagent tray, a cooler, and a position sensor that enables movement of the adjustable stage to enable selection of reagents from the reagent tray.In some embodiments, the fluid system further comprises a waste container fluidly connected to a multi-directional 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 widefield microscopy. In some embodiments, the fluid system further comprises a container surrounding the fluid system, the container blocking ambient light. In some embodiments. In some embodiments, the container surrounding the fluid system comprises one or more doors or drawers. In some embodiments, the container surrounding the fluid system is an acoustic attenuation container.

Brief Description of the Drawings

[0008]

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DETAILED DESCRIPTION OF THE INVENTION

[0009] Devices, methods, and systems are provided herein for in situ gene sequencing of target nucleic acids in cells in a tissue in vivo. Methods are also provided herein for screening a candidate agent to determine whether the candidate agent modulates gene expression of a nucleic acid in cells in a tissue in vivo.

[0010] Prior to describing the device, method, and system, it is to be understood that the invention is not limited to the specific methods or compositions described, as such may, of course, vary. Also, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the invention is defined only by the appended claims.

[0011] Where a range of values is provided, it is to be understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range is specifically disclosed herein, unless the context clearly dictates otherwise. Each smaller range between any of the recited values or intervening values in a recited range and any other recited value or intervening value in that recited range is also contemplated by the invention. The upper and lower limits of these smaller ranges may independently be included in or excluded from the range, and ranges encompassing any one, neither, or both of the limits are also contemplated by the invention, subject to any specifically excluded limit in the recited range. Where the recited range includes one or both of the limits, ranges excluding either or both of those included limits are also included in 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. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but some potential and preferred methods and materials are described herein. All publications mentioned herein are incorporated herein by reference for the purpose of disclosing and describing the methods and / or materials in connection with which the publications are cited. It is to be understood that, in case of any conflict, the present disclosure prevails over 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 can be readily separated from, or combined with, any of the features of any of the other various embodiments without departing from the scope or spirit of the present invention. Any recited method can be performed in the order of the recited events, or in any other logically possible order.

[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, reference to "a cell" includes a plurality of such cells, and reference to "the peptide" includes reference to one or more peptides known to those skilled in the art and their equivalents, such as oligopeptides or polypeptides.

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

[0016] Definitions The term "about" means including 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 and refer to a polymer of amino acid residues. This term also applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. Both full-length proteins and fragments thereof are included in the definition. This 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. This term also includes fusion proteins having heterologous amino acid sequences, fusions having heterologous and homologous leader sequences with or without an N-terminal methionine residue, immunologically tagged proteins, etc., including but not limited to fusion proteins. This 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" is any polynucleotide nucleic acid molecule (e.g., a DNA molecule, an RNA molecule, a 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 cellular context. Thus, suitable target nucleic acids are unspliced RNAs (e.g., pre-mRNA, mRNA), partially spliced It can be, for example, circular RNA, or fully spliced RNA. The target nucleic acid of interest may be variably expressed within a cell population, i.e., may have different abundances, and the methods of the present invention enable 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, for example, a denatured genome, virus, plasmid, etc. For example, the method can be used to detect copy number variants in, for example, cancer cell populations where the target nucleic acid is present in different abundances in the genomes of cells in the population, virus-infected cells for determining virus load and kinetics.

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

[0020] When referring to a "isolated" protein, polypeptide, or peptide, it means that the indicated molecule is separated from and is distinct from the whole organism in which the molecule is found in nature, or is present in the substantial absence of other biological macromolecules of the same type. The term "isolated" with respect to a polynucleotide refers to a nucleic acid molecule that lacks all or part of the sequences that are normally associated with it in nature, or has sequences that are naturally occurring but are associated with heterologous sequences sequences, or is a molecule dissociated from a chromosome.

[0021] The terms "subject", "individual", or "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal. "Vertebrate" means any member of the subphylum Vertebrata, including humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species, other primates, livestock such as cows, sheep, pigs, goats and horses, domesticated mammals such as dogs and cats, laboratory animals including rodents such as mice, rats, and guinea pigs, birds including chickens, turkeys and other poultry birds, domesticated birds such as ducks, geese, wild birds and game birds, but not limited to these. The term does not indicate a particular age. Thus, both adult and neonatal individuals are intended to be covered.

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

[0023] As summarized above, the methods disclosed herein include methods for in situ gene sequencing of target nucleic acids in cells in intact tissue. The method typically includes (a) contacting an immobilized and permeabilized intact tissue sample with at least a pair of oligonucleotides under conditions that allow specific hybridization, wherein the pair of oligonucleotides includes a first oligonucleotide and a second oligonucleotide, each of the first oligonucleotide and the second oligonucleotide includes a first complementary region, a second complementary region, and a third complementary region, the second oligonucleotide further includes a barcode sequence, 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, the second complementary region of the second oligonucleotide is complementary to a second portion of the target nucleic acid, and 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 generate a closed nucleic acid ring; (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) contacting the tissue sample with deoxyribonucleotide triphosphates such that one or more amplicons are generated by rolling circle amplification. Performing circular amplification, wherein the ligated second oligonucleotide functions as a template and the first oligonucleotide functions as a primer for DNA polymerase; (e) embedding a tissue sample in a hydrogel before or after any one of steps (a) to (d); (f) crosslinking one or more amplicons to the 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 bound to a fluorophore comprising a second thiol group by a disulfide bond between the first thiol group and the second thiol group; (i) ligating the lead primer and the fluorescently labeled probe, wherein ligation occurs only when 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-fading buffer containing an antioxidant; (k) imaging one or more hydrogel-embedded amplicons to determine the positioning of the target nucleic acid in cells in intact tissue undergoing in situ gene sequencing, wherein imaging is performed in the presence of the anti-fading buffer; (l) contacting the hydrogel with a reducing agent to effect reduction of the disulfide bond and cleavage of the fluorophore from the probe; (m) removing the fluorophore from the hydrogel; and (n) repeating steps (h) to (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 intact tissue, the method comprising: (a) contacting an immobilized 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 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, the third complementary region of the first oligonucleotide being complementary to the third complementary region of the second 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 second oligonucleotide to generate a closed nucleic acid ring; (c) performing rolling circle amplification in the presence of a nucleic acid molecule, using the second oligonucleotide as a template and the first oligonucleotide as a primer for a polymerase to form one or more amplicons; (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 a barcode sequence with a pair of primers under conditions that allow ligation, the pair of primers comprising a third oligonucleotide and a fourth oligonucleotide, the ligation occurring only when both the third oligonucleotide and the fourth oligonucleotide ligate to the same amplicon; and (f) repeating step (e).(g) Imaging one or more hydrogel-embedded amplicons to determine in situ gene sequencing of target nucleic acids in cells in intact tissue, and (h) detecting the gene expression level of the target nucleic acid, wherein the change in the expression level of the target nucleic acid in the presence of at least one candidate agent relative to the expression level of the target nucleic acid in the absence of at least one candidate agent indicates that at least one candidate agent regulates the gene expression of nucleic acids in cells in intact tissue. including detecting, as shown.

[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 a hydrogel imprinted with nucleic acids compatible with in situ sequencing, a ligation-based sequencing 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 infections, viral infections, 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 includes an efficient approach for generating a cDNA library 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 methods include contacting fixed and permeabilized intact tissue with at least a pair of oligonucleotide primers under conditions that permit specific hybridization, the pair of primers including a first oligonucleotide and a second oligonucleotide.

[0027] More generally, nucleic acids present in target cells in an organization function as a scaffold for the association of a complex containing a pair of primers, referred to herein as a first oligonucleotide and a second oligonucleotide. In some embodiments, contacting the immobilized and permeabilized intact tissue involves 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 via Watson-Crick base pairing. When a primer "hybridizes" to a target (template), such a complex (or hybrid) is sufficiently stable to perform the priming function required, for example, by a DNA polymerase to initiate DNA synthesis. It is understood that the hybridizing sequences need not have perfect complementarity to provide a stable hybrid. In many cases, loops of four or more nucleotides are ignored and less than about 10% of the bases are mismatched and a stable hybrid is formed. Thus, as used herein, the term "complementary" generally refers to an oligonucleotide that forms a stable double strand with its "complement" under assay conditions where there is at least about 90% homology.

[0029] SNAIL oligonucleotide primer 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, the third complementary region of the first oligonucleotide being complementary to the third complementary region of the second 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. 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 that includes hybridizing a plurality of oligonucleotide primers having specificity for different target nucleic acids with immobilized and permeabilized tissue. In some embodiments, the method includes a plurality of first oligonucleotides that hybridize to a target nucleotide sequence, including, but not limited to, 5 or more first oligonucleotides, such as 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 includes a plurality of first oligonucleotides that hybridize to 15 or more, such as 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, including, but not limited to, 15 or more first oligonucleotides, such as 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 second oligonucleotides that include, but are not limited to, 5 or more second oligonucleotides, such as 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 includes a plurality of second oligonucleotides that hybridize to 15 or more, such as 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, including, but not limited to, 15 or more second oligonucleotides, such as 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. A plurality of oligonucleotide pairs can be used in the reaction, and one or more pairs specifically bind to each target nucleic acid. For example, two primer pairs can be used for one target nucleic acid to improve sensitivity and reduce variability.It is also an object to detect a plurality of different target nucleic acids in a cell, for example, to detect 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 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 before use.

[0031] In some embodiments, the primer is denatured by heating before contacting the sample. In certain embodiments, the melting temperature (T m ) of the oligonucleotide is selected to minimize ligation in solution. The "melting temperature" or "T m " of a nucleic acid is defined as the temperature at which half of the helical structure of the nucleic acid is lost due to heating or other dissociation of hydrogen bonds between base pairs, such as by acid or alkali treatment. The T m of a nucleic acid molecule depends on its length and base composition. Nucleic acid molecules rich in GC base pairs have a higher T m than those rich in AT base pairs. The complementary strands of a separated nucleic acid will spontaneously reassociate or anneal to form double-stranded nucleic acid when the temperature drops below T m . The highest rate of nucleic acid hybridization occurs approximately 25°C below T m . 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 include 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 detectable molecules including, but not limited to, radioisotopes, phosphors, chemiluminescent agents, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin or hapten), and the like. The term "fluorescent agent" refers to a substance or a part thereof that can exhibit fluorescence within a detectable range. Specific examples of labels that can be used with the present invention include phycoerythrin, Alexa dyes, fluorescein, YPet, CyPet, cascade blue, allophycocyanin, Cy3, Cy5, Cy7, rhodamine, dansyl, umbelliferone, Texas red, luminol, acridinium ester, 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 urease, but are not limited thereto.

[0033] In some embodiments, one or more first oligonucleotides and second oligonucleotides bind to different regions of a target nucleic acid or target site. In a pair, each target site is different, and the target sites are adjacent sites on the target nucleic acid, for example, usually at a distance of 15 nucleotides or less from other sites, for example, at a distance of 10, 8, 6, 4, or 2 nucleotides or less, and can be contiguous sites. The target sites typically exist on the same strand of the target nucleic acid in the same direction. The target sites are also selected to provide unique binding sites for other nucleic acids present within the cell. Each target site is generally about 19 to about 25 nucleotides in length, for example, about 19 to 23 nucleotides, about 19 to 21 nucleotides, or about 19 to 20 nucleotides. The pair of first and second oligonucleotides is selected such that each oligonucleotide in the pair has a similar melting temperature for binding to its cognate target site, for example, 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 first, second, and third complementary regions. 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 can have a length of 19 to 25 nucleotides. In certain embodiments, the second complementary region of the first oligonucleotide has a length of 3 to 10 nucleotides, for example, 4 to 8 nucleotides or 4 to 7 nucleotides. In some embodiments, 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, for example, 4 to 8 nucleotides or 4 to 7 nucleotides.

[0035] In some embodiments, the second oligonucleotide comprises first, second, and third complementary regions. 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 embodiments, 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 embodiments, the first complementary region of the first oligonucleotide has a length of 6 nucleotides. In some embodiments, the first complementary region of the second oligonucleotide includes 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 includes 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 barcoding information for identification of the target nucleic acid. The term "barcode" refers to a nucleic acid sequence used to identify a single cell or subpopulation of cells. The barcode sequence can be ligated to the target nucleic acid of interest during amplification and used to trace the amplicon back to the cell from which the target nucleic acid is derived. The barcode sequence can be added to the target nucleic acid of interest during amplification by performing amplification with an oligonucleotide containing a region that includes the barcode sequence and a region complementary to the target nucleic acid such that the barcode sequence is incorporated into the target nucleic acid product (i.e., the amplicon) that is ultimately amplified.

[0038] tissue As described herein, the disclosed method includes an in situ sequencing technique by contacting at least a pair of oligonucleotide primers with intact tissue that has been immobilized and permeabilized under conditions that allow specific hybridization. Suitable tissue specimens for use with the methods described herein generally include, but are not limited to, biopsy and autopsy specimens such as epithelial, muscle, connective, and nervous tissues, and any type of tissue specimen collected from a living or dead subject, such as those. The tissue specimen may be collected and processed using the methods described herein, subjected to microscopic analysis immediately after processing, or stored and, in the future, subjected to microscopic analysis, for example, after long-term storage. In some embodiments, the methods described herein can be used to store tissue specimens in a stable, accessible, and fully intact form for future analysis. In some embodiments, the methods described herein can be used to analyze previously stored or archived tissue specimens. In some embodiments, the intact tissue includes brain tissue such as visual field slices. In some embodiments, the intact tissue is a thin slice having a thickness of 5-20 μm, including, but not limited to, 5-18 μm, 5-15 μm, or 5-10 μm. In other embodiments, the intact tissue is a thick slice having a thickness of 50-200 μm, including, but not limited to, 50-150 μm, 50-100 μm, or 50-80 μm.

[0039] Aspects of the present invention include immobilizing intact tissue. As used herein, the terms "immobilize" or "immobilizing" refer to the process of preserving biological materials (e.g., tissue, cells, organelles, molecules, etc.) from decay and / or degradation. Immobilization can be achieved using any convenient protocol. Immobilization can include contacting the sample with an immobilization reagent (i.e., a reagent containing at least one immobilizing agent). The sample can be contacted with the immobilization reagent for a wide range of times, which can depend on the temperature, the nature of the sample, and the immobilizing agent. For example, the sample can be contacted with the immobilization 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 can be contacted with the immobilization reagent for a period ranging from 5 minutes to 24 hours, such as 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 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 can be contacted with the immobilization reagent at various temperatures, depending on the protocol and the reagent used. For example, in some instances, the sample can be contacted with the immobilization reagent at a temperature in the range of -22°C to 55°C, and specific ranges of interest include, but are not limited to, 50 - 54°C, 40 - 44°C, 35 - 39°C, 28 - 32°C, 20 - 26°C, 0 - 6°C, and -18 - -22°C. In some cases, the sample can be contacted with the immobilization reagent at a temperature of -20°C, 4°C, room temperature (22 - 25°C), 30°C, 37°C, 42°C, or 52°C.

[0042] Any convenient immobilization reagent can be used. Common immobilization reagents include crosslinking immobilizing agents, precipitation immobilizing agents, oxidation immobilizing agents, mercury, and the like. Crosslinking immobilizing agents chemically bond two or more molecules by covalent bonds, and a wide range of crosslinking reagents can be used. Examples of suitable crosslinking immobilizing agents include aldehydes (e.g., formaldehyde, also commonly referred to as "paraformaldehyde" and "formalin", glutaraldehyde, etc.), imide esters, NHS (N-hydroxysuccinimide) esters, etc., but are not limited thereto. Examples of suitable precipitation immobilizing agents include alcohols (e.g., methanol, ethanol, etc.), acetone, acetic acid, etc., but are not limited thereto. In some embodiments, the immobilizing agent is formaldehyde (i.e., paraformaldehyde or formalin). Suitable final concentrations of formaldehyde in the immobilization reagent are 0.1 - 10%, 1 - 8%, 1 - 4%, 1 - 2%, 3 - 5%, or 3.5 - 4.5% including about 1.6% in 10 minutes. In some embodiments, the sample is 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, the sample is immobilized at a final concentration of 10% formaldehyde. In some embodiments, the sample is immobilized at a final concentration of 1% formaldehyde. In some embodiments, the immobilizing agent is glutaraldehyde. A suitable concentration of glutaraldehyde in the immobilization reagent is 0.1 - 1%. The immobilization reagent may contain two or more immobilizing agents in any combination. For example, in some embodiments, the sample is contacted with an immobilization reagent containing both formaldehyde and glutaraldehyde.

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

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

[0045] The sample can be contacted with the permeabilization reagent for a wide range of times that can depend on the temperature, the nature of the sample, and the permeabilization reagent. For example, the sample can 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 can be contacted with the permeabilization reagent at various temperatures depending on the protocol and the reagent used. For example, in some instances, the sample can be contacted with the permeabilization reagent at a temperature in the range of -82°C to 55°C, and specific ranges of interest include, but are not limited to, 50 - 54°C, 40 - 44°C, 35 - 39°C, 28 - 32°C, 20 - 26°C, 0 - 6°C, -18 - -22°C, and -78 - -82°C. In some instances, the sample can 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 enzymatic permeabilization reagent. The enzymatic permeabilization reagent permeabilizes the sample by partially degrading extracellular matrix or surface proteins that impede permeabilization of the sample by the assay reagent. Contact with the enzymatic permeabilization reagent can occur at any point after fixation and before target detection. In some examples, the enzymatic permeabilization reagent is Proteinase K, a commercially available enzyme. In such cases, the sample is contacted with Proteinase K prior to contacting with the reagent after immobilization. Proteinase K treatment (i.e., contact with Proteinase K, generally also referred to as “Proteinase K digestion”) can be carried out over a range of temperatures and over a range of times over a range of enzyme concentrations empirically determined for each cell type 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 can 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 can be contacted with Proteinase K at a temperature in the range of 2° C. to 55° C., and specific ranges of interest include, but are 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 can 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 enzymatic permeabilization reagent. In some embodiments, the sample is not contacted with Proteinase K. Contact of the intact tissue with at least the immobilization reagent and the permeabilization reagent results in the production of an immobilized and permeabilized tissue.

[0047] Ligase In some embodiments, the disclosed method includes ligating a second oligonucleotide and adding a ligase to generate a closed nucleic acid ring. In some embodiments, the addition of the ligase includes the addition of a DNA ligase. In alternative embodiments, the second oligonucleotide is provided as a closed nucleic acid ring, and the step of adding a ligase is omitted. In certain embodiments, the ligase is an enzyme that facilitates the sequencing of a 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 those described in EC6.5.1.1 (ATP-dependent ligase), EC6.5.1.2 (NAD+-dependent ligase), EC6.5.1.3 (RNA ligase). Specific examples of ligases include bacterial ligases such as E. coli DNA ligase and Taq DNA ligase, Ampligase® thermostable DNA li gase (Epicentre® Technologies Corp., a 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 invention includes performing rolling circle amplification in the presence of a nucleic acid molecule, which includes using a second oligonucleotide as a template and a first oligonucleotide as a primer for a polymerase to form one or more amplicons. In such embodiments, the single-stranded circular polynucleotide template is formed by ligation of the second nucleotide, and the circular polynucleotide includes a region complementary to the first oligonucleotide. When a DNA polymerase is added in the presence of appropriate dNTP precursors and other cofactors, the first oligonucleotide is extended by replication of multiple copies of the template. This amplification product can be easily detected by binding to a detection probe.

[0050] In some embodiments, the second oligonucleotide can be circularized and rolling circle amplified to generate cDNA nanoballs (i.e., amplicons) containing multiple copies of cDNA only when the first oligonucleotide and the second oligonucleotide hybridize to the same target nucleic acid molecule. The term "amplicon" refers to an 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 (see al, BMC Genomics 2:4, 2000, Nallur et al, Nucl. Acids Res. 29:el18, 2001, Dean et al. Genome Res. 11:1095-1099, 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 acrylate N-hydroxysuccinimide moiety modification. Examples of other amine-modified nucleotides include, but are not limited to, 5-aminoallyl-dUTP moiety modification, 5-propynylamino-dCTP moiety modification, N6-6-aminohexyl-dATP moiety modification, or 7-deaza-7-propynylamino-dATP moiety modification.

[0053] Setting of amplicon embedding in tissue hydrogel In some embodiments, the disclosed method comprises embedding one or more amplicons in the presence of a hydrogel subunit 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 purification, enzyme diffusion, and sequencing. In some embodiments, to enable the setting of amplicon embedding in tissue-hydrogel, amine-modified nucleotides are spiked into a rolling circle amplification reaction and functionalized with acrylate N-hydroxysuccinimide ester is used to functionalize with acrylamide moieties and copolymerize with acrylamide monomers to form a hydrogel.

[0054] As used herein, the terms "hydrogel" or "hydrogel network" mean a network of water-insoluble polymer chains, sometimes found as a colloidal gel in which water is the dispersion medium. In other words, a hydrogel is a class of polymeric materials that can absorb large amounts of water without dissolving. Hydrogels can contain over 99% water and can include natural polymers, synthetic polymers, or combinations thereof. Hydrogels also have a degree of flexibility very similar to natural tissues due to their significant water content. A detailed description of suitable hydrogels can be found in U.S. Patent Application No. 2010 / 0055733, which is specifically incorporated herein by reference. As used herein, the terms "hydrogel subunit" or "hydrogel precursor" mean hydrophilic monomers, prepolymers, or polymers that can be crosslinked or "polymerized" to form a three-dimensional (3D) hydrogel network. Without being bound by any scientific theory, this fixation of a biological sample in the presence of hydrogel subunits is thought to crosslink the components of the specimen to the hydrogel subunits, thereby fixing the molecular components in place and preserving tissue structure and cell 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 embodiments, encapsulation includes purifying one or more hydrogel-encapsulated amplicons, and the target nucleic acid is substantially retained within the one or more hydrogel-encapsulated amplicons. In such embodiments, purification includes substantially removing a plurality of cellular components from the one or more hydrogel-encapsulated amplicons. In some other embodiments, purification includes substantially removing lipids from the one or more hydrogel-encapsulated amplicons. As used herein, the term "substantially" means that the original amount present in the sample prior to purification has been reduced by approximately 70% or more, 75% or more, such as 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, 100%, etc.

[0057] In some embodiments, purification of the hydrogel-encapsulated amplicons includes electrophoresing the sample. In some embodiments, the amplicons are electrophoresed using a buffer solution containing an ionic surfactant. In some embodiments, the ionic surfactant is sodium dodecyl sulfate (SDS). In some embodiments, the sample is electrophoresed using a voltage in the range of about 10 to about 60 volts. In some embodiments, the sample is electrophoresed for a period ranging from about 15 minutes to a maximum of about 10 days. In some embodiments, the method further includes incubating the purified sample in an encapsulating medium having a refractive index that matches the refractive index of the purified tissue. In some embodiments, the encapsulating medium increases the optical transparency of the sample. In some embodiments, the encapsulating medium contains glycerol.

[0058] Sequencing Error Correction by Dynamic Annealing and Ligation (SEDAL) The methods disclosed herein include contacting one or more hydrogel-embedded amplicons having barcode sequences with a pair of primers under conditions that permit 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 ligation-based sequencing method is performed at room temperature for preservation of tissue morphology, has low background noise and error reduction. In some embodiments, contacting the one or more hydrogel-embedded amplicons includes eliminating the accumulation of errors as sequencing proceeds.

[0059] In some embodiments, contacting the one or more hydrogel-embedded amplicons occurs two or more times, including, but not limited to, 3 or more times, 4 or more times, 5 or more times, 6 or more times, or 7 or more times. In certain embodiments, contacting the one or more hydrogel-embedded amplicons occurs 4 or more times for thin tissue specimens. In other embodiments, contacting the one or more hydrogel-embedded amplicons occurs 6 or more times for thick tissue specimens. In some embodiments, one or more amplicons can be contacted with the pair of primers 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.

[0060] Specimens prepared using the subject method 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 reflection, epifluorescence, confocal microscopy, spinning disk confocal microscopy), light sheet microscopy, lattice light sheet microscopy, 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 obtained via multiple rounds of in situ array determination microscopy into decoded gene identities and spatial positions and then analyzes the per-cell composition of gene expression.

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

[0062] The term "fully complementary" when used with respect to a double strand means that the polynucleotide strand and / or oligonucleotide strand that makes up the double strand form a double-stranded structure with each other such that all nucleotides in each strand undergo Watson-Crick base pairing with nucleotides in the other strand. The term "double strand" includes, but is not limited to, nucleoside analogs such as deoxyinosine, nucleosides having 2-aminopurine bases, and pairings such as peptide nucleic acids (PNAs), and they can be used. "Mismatch" in a 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 method includes a plurality of third oligonucleotides that hybridize to a target nucleotide sequence, including, but not limited to, 5 or more third oligonucleotides, such as 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 methods of the disclosure include 15 or more third oligonucleotides that hybridize to 15 or more, such as 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. A plurality of third oligonucleotides, comprising nucleotides, 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 first oligonucleotides. In some embodiments, the method includes a plurality of fourth oligonucleotides, including but not limited to 5 or more, 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. In some embodiments, the method of the present disclosure includes 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 fourth oligonucleotides that hybridize 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, including a plurality of fourth oligonucleotides, including but not limited to 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. A plurality of oligonucleotide pairs can be used in the reaction, and one or more pairs specifically bind to each target nucleic acid. For example, two primer pairs can be used for one target nucleic acid to improve sensitivity and reduce variability. It is also an object to detect a plurality of different target nucleic acids in a cell, e.g., 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, SEDAL includes a ligase having an activity that is inhibited by a base mismatch, a third oligonucleotide, and a fourth oligonucleotide. The term "inhibited" in this context refers to a reduction in ligase activity of about 20% or more, 25% or more, such as 50% or more, 75% or more, 90% or more, 95% or more, 99% or more, 100%, etc. 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 m of the third oligonucleotide 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 m of the fourth oligonucleotide is room temperature (22-25 °C). After each cycle of SEDAL corresponding to a base read, the fourth oligonucleotide may be stripped, thereby eliminating error accumulation as sequencing progresses. In such embodiments, the fourth oligonucleotide is stripped by formamide.

[0065] In some embodiments, SEDAL involves washing a third and a fourth oligonucleotide to eliminate unbound oligonucleotides, and then revealing the fluorescent products for imaging. In certain exemplary embodiments, a detectable label can be used to detect one or more nucleotides and / or the oligonucleotides described herein. In certain embodiments, a detectable label can be used to detect one or more amplicons. Examples of detectable markers include various radioactive moieties, enzymes, haptens, 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, luciferase (e.g., bacteria, firefly, glow worm, etc.), luciferin, aequorin, etc. Enzymes with a visually detectable signal Examples of systems include, but are not limited to, galactosidase, glucuronidase, phosphatase, peroxidase, cholinesterase, etc. Identifiable markers include 125 I, 35 S, 14 C, or 3 radioactive compounds such as H, etc. Identifiable markers are commercially available from various sources.

[0066] The binding of fluorescent labels and their nucleotides and / or oligonucleotides is described in Haugland, Handbook of Fluorescent Probes and Research Chemicals, Ninth Edition (Molecular Probes, Inc., Eugene, 2002), Keller and Manak, DNA Probes, 2nd Edition (Stockton Press, New York, 1993), Eckstein, editor, Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991) and many reviews including Wetmur, Critical Reviews in Biochemistry and Molecular Biology, 26:227-259 (1991). Specific methodologies applicable to the present invention are disclosed in the reference examples of U.S. Patent Nos. 4,757,141, 5,151,507, and 5,091,519. In one aspect, one or more fluorescent dyes are used as labels for labeled target sequences as disclosed, for example, in U.S. Patent No. 5,188,934 (4,7-dichlorofluorescein dye), U.S. Patent No. 5,366,860 (spectrally resolvable rhodamine dye), U.S. Patent No. 5,847,162 (4,7-dichlororhodamine dye), U.S. Patent No. 4,318,846 (ether-substituted fluorescein dye), U.S. Patent No. 5,800,996 (energy transfer dye), and Lee et al., U.S. Patent No. 5,066,580 (xanthine dye), U.S. Patent No. 5,688,648 (energy transfer dye), etc. Labeling can also be performed using quantum dots as disclosed in the patents and patent publications of 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 a signal transduction moiety that transmits information through the fluorescence absorption and / or emission characteristics of one or more molecules. Such fluorescent characteristics include fluorescence intensity, fluorescence lifetime, emission spectral characteristics, energy transfer, etc.

[0067] Commercially available fluorescent nucleotide analogs that can be easily incorporated into nucleotide and / or oligonucleotide sequences include Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dUTP (Amersham Biosciences, Piscataway, N.J.), fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, TEXAS RED™-5-dUTP, CASCADE BLUE™-7-dUTP, BODIPY™ FL-14-dUTP, BODIPY™ TMR-14-dUTP, BODIPY™ TR-14-dUTP, RHODAMINE GREEN™-5-dUTP, OREGON GREEN®488-5-dUTP, TEXAS RED™-12-dUTP, BODIPY™ 630 / 650-14-dUTP, BODIPY™ 650 / 665-14-dUTP, ALEXA FLUOR®488-5-dUTP, ALEXA FLUOR®532-5-dUTP, ALEXA FLUOR®568-5-dUTP, ALEXA FLUOR®594-5-dUTP, ALEXA FLUOR®546-14-dUTP, fluorescein-12-UTP, tetramethylrhodamine-6-UTP, TEXAS RED™-5-UTP, mCherry, CASCADE BLUE™-7-UTP, BODIPY™ FL-14-UTP, BODIPY™ 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.), and the like, but are not limited thereto. Protocols for custom synthesis of nucleotides having other fluorophores are known in the art (see Henegariu et al. (2000) Nature Biotechnol. 18:345).

[0068] Other fluorophores available for use in the post-synthesis conjugation include, but are not limited to, 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, Pacific Blue, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Tetramethylrhodamine, Texas Red (available from Molecular Probes, Inc., Eugene, Oreg.), Cy2, Cy3.5, Cy5.5, Cy7 (Amersham Biosciences, Piscataway, N.J.). FRET tandem fluorophores can also be used and include, but are 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] Metal silver or gold particles can be used to enhance the signal from fluorescently labeled nucleotides and / or oligonucleotide sequences (Lakowicz et al. (2003) Bio Techniques 34:62).

[0070] Biotin or its derivatives can be used as labels on nucleotide and / or oligonucleotide sequences, and can then be bound by 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 and can then be bound by a detectably labeled anti-digoxigenin antibody (e.g., fluoresceinylated anti-digoxigenin). Aminoallyl-dUTP residues can be incorporated into an oligonucleotide sequence and subsequently bound to an N-hydroxysuccinimide (NHS)-derivatized fluorescent dye. Generally, any member of a conjugate pair can be incorporated into a detection oligonucleotide, provided that a detectably labeled conjugate partner can be bound to enable detection. As used herein, the term antibody refers to any class of antibody molecule, or any sub-fragment thereof such as Fab.

[0071] Other suitable labels for oligonucleotide sequences can include fluorescein (FAM), digoxigenin, dinitrophenol (DNP), dansyl, biotin, bromodeoxyuridine (BrdU), hexahistidine (6×His), phosphorescent amino acids (e.g., P-tyr, P-ser, P-thr), and the like. In one embodiment, the following hapten / antibody pairs are used for detection, and each of the antibodies is 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, for example, with haptens that are then bound by a capture agent, as disclosed in, for example, 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, and the like. Many different hapten capture agent pairs can be used. Exemplary haptens include, but are not limited to, biotin, desthiobiotin and other derivatives, dinitrophenol, dansyl, fluorescein, CY5, digoxigenin, and the like. 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 tissue. In certain embodiments, the cells are present in a population of cells. In certain other embodiments, the population of cells includes 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 a biological organism, a single cell type derived from a biological organism, or a mixture of cell types. Included are naturally occurring cells and cell populations, genetically engineered cell lines, cells derived from transgenic animals, and the like. Virtually any cell type and size can be accommodated. Suitable cells include bacterial, fungal, plant, and animal cells. In one embodiment of the invention, the cells are mammalian cells, for example, a complex population of cells from a naturally occurring tissue such as blood, liver, pancreas, nervous tissue, bone marrow, skin, and the like. Some tissues can be disrupted into a single - cell suspension. Alternatively, the cells can be a cultured population, for example, a culture derived from a complex population, a single cell type in which the cells have differentiated into multiple lineages, or a culture in which the cells respond differentially to a stimulus.

[0074] Cell types that can be found for use in the claimed invention include stem cells and progenitor cells, such as embryonic stem cells, hematopoietic stem cells, mesenchymal stem cells, neural crest cells, etc., endothelial cells, muscle cells, cardiac muscle, smooth muscle and skeletal muscle cells, mesenchymal cells, epithelial cells, lymphocytes including T cells such as Th1 T cells, Th2 T cells, ThO T cells, cytotoxic T cells, etc., B cells, pre-B cells, etc., monocytes, dendritic cells, neutrophils, and macrophages, natural killer cells, mast cells, etc., adipocytes, thymus, endocrine glands, pancreas, neurons, glia, astrocytes, cells involved in specific organs such as the brain including dendritic cells, and their genetically engineered cells, etc. Hematopoietic cells may be associated with inflammatory processes, autoimmune diseases, etc., and endothelial cells, smooth muscle cells, cardiac muscle cells, etc. may be associated with cardiovascular diseases. Almost any type of cell may be associated with tumors such as sarcomas, cancers and lymphomas, liver diseases with hepatocytes, kidney diseases with renal cells, etc.

[0075] Cells may also be different types of transformed cells or tumor cells, such as carcinomas of different cell origins, lymphomas of different cell types, etc. The American Type Culture Collection (Manassas, VA) has collected and made available over 4,000 cell lines from over 150 different species, including over 950 cancer cell lines including 700 human cancer cell lines. The National Cancer Institute has compiled 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. (1996) Journal of Cellular Biochemistry Supplement 24:32 - 91). Different cell lines, either spontaneously derived or selected for desired growth or response characteristics from individual cell lines, are included, and multiple cell lines derived from a similar tumor type but from different patients or sites may be included.

[0076] The cells may be non-adherent, for example, blood cells including monocytes, T cells, B cells, tumor cells, etc., or adherent cells such as epithelial cells, endothelial cells, nerve cells, etc. In order to profile adherent cells, they may be dissociated from the substrate to which they adhere and from other cells in a manner that maintains the ability to recognize and bind probe molecules.

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

[0078] Imaging The disclosed method involves imaging one or more hydrogel-embedded amplicons using any of several different types of microscopy, such as confocal microscopy, two-photon microscopy, brightfield microscopy, label-free tissue expansion microscopy, and / or CLARITY (trademark) optimized light sheet microscopy (COLM).

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

[0080] In an oblique illumination microscope, the specimen is illuminated from the side. This gives the image a three-dimensional appearance and can highlight features that would otherwise not be visible. A more recent technique based on this method is Hoffmann modulation contrast, a system found in the inverted microscopes used for cell culture. Oblique illumination suffers from the same limitations as brightfield microscopy (low contrast of many biological specimens, apparent low resolution due to out-of-focus objects), but can highlight structures that would otherwise not be visible.

[0081] Darkfield microscopy is a technique for improving the contrast of unstained transparent specimens. Darkfield illumination uses a carefully aligned light source to minimize the amount of direct transmitted (non-scattered) light entering the image plane and collect only the light scattered by the specimen. Darkfield can dramatically improve the contrast of an image (especially of transparent objects) with little need for equipment setup and specimen preparation. However, this technique suffers from low light intensity in the final images of many biological specimens and continues to be affected by apparent low 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 shows differences in refractive index as differences in contrast. The phase shift itself is not visible 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 special prisms (Nomarski prism, Wollaston prism) in the condenser that split the light of an ordinary beam and an extraordinary beam. The spatial difference between the two beams is minimal (less than the maximum resolution of the objective lens). After passing through the specimen, the beams are recombined by similar prisms within the objective lens. In a homogeneous specimen, 2 There is no difference between the two beams and no contrast is generated. However, near a refractive boundary (e.g., the nucleus within the cytoplasm), the difference between the normal beam and the extraordinary beam causes relaxation in the image. Differential interference contrast requires a polarizer to function and two polarizing filters need to be attached to the optical path, one under the condenser (polarizer) and the other above the objective lens (analyzer).

[0084] Another microscopy technique that uses interference is interference reflection microscopy (also known as reflected interference contrast, or RIC). This is used to examine the adhesion of cells to a glass surface by using polarized light in a narrow range of wavelengths that is always reflected when there is an interface between two substances with different refractive indices. When a cell binds to the glass surface, the reflected light from the glass interferes with the reflected light from the cell that has bound to it. If there is no cell bound to the glass, there is no interference.

[0085] A fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence to study the properties of organic or inorganic substances, instead of or in addition to reflection and absorption. In fluorescence microscopy, the sample is irradiated with light of a wavelength that excites the fluorescence in the sample. The fluorescence light, which is usually at a longer wavelength than the illumination, is then imaged through the microscope objective lens. In this technique, two filters can be used. An illumination (or excitation) filter that ensures the illumination is near-monochromatic and at the correct wavelength, and a second emission (or barrier) filter that 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 generate an image, whether it is a simpler setup like a fluorescence microscope or a more complex design like a confocal microscope that uses optical sections to obtain better resolution of the fluorescence image.

[0086] In some embodiments, the antioxidant compound is included in a wash and imaging buffer (i.e., an "antifade buffer") 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 antifade effect on fluorophores. That is, the antioxidant reduces photobleaching during tiling, significantly enhances the SNR of sensitive fluorophores, and enables higher SNR imaging of thicker samples. In the case of a fixed exposure time containing an antioxidant, the SNR is increased by increasing the concentration of the phosphor that has not been bleached during exposure to light. By including an antioxidant, the reduction in return of longer exposure times (caused by the limited fluorescence lifetime before photobleaching) is also removed, providing an increase in SNR by allowing an increase in exposure time.

[0087] In addition, fluorophore cleavage from the probe (as opposed to probe stripping) is used in the CISITS method to eliminate signal from round to round. By including a disulfide bond between the fluorophore and the oligonucleotide probe, cleavage of the fluorophore from the oligonucleotide probe in a reducing environment is enabled. After fluorescence imaging of the sequencing round, a reducing agent is added to reduce the disulfide bond connecting the fluorophore to the oligonucleotide, and a subsequent wash step removes the diffusible fluorescent signal before another round is performed. By using SEDAL sequencing with thiol-based chemical cleavage in CISITS, the specificity and SNR advantages of SEDAL sequencing are combined with the robustness and rapidity of the round-to-round cycle from fluorophore cleavage.

[0088] A confocal microscope uses point illumination and a pinhole in an optically conjugate plane in front of the detector to exclude out-of-focus signals. Only light generated by fluorescence very close to the focal plane is detected. Since it cannot come out, the optical resolution of the image, especially in the sample depth direction, is much better than that of a wide-field microscope. However, since much of the light from the sample fluorescence is blocked by the pinhole, this increased resolution comes at the expense of a decrease in signal intensity, and therefore long exposure times are often required. Since only one point in the sample is irradiated at a time, 2D imaging or 3D imaging needs to be scanned against a regular raster in the specimen (i.e., a rectangular pattern of parallel scanning lines). The achievable thickness of the focal plane is defined mainly by dividing the wavelength of the light used by the numerical aperture of the objective lens, but also by the optical properties of the specimen. With thin optical sectioning, these types of microscopes may be particularly excellent for 3D imaging and surface profiling of samples. COLM provides an alternative microscope for high-speed 3D imaging of large cleared samples. COLM examines large immunostained tissues, enables an increase in collection speed, and results in higher quality of the generated data.

[0089] In single plane illumination microscopy (SPIM), also known as light sheet microscopy, only the fluorophores in the focal plane of the detection objective are irradiated. The light sheet is a beam that is parallelized in one direction and converged in the other direction. Since fluorophores are not excited outside the focal plane of the detector, the method also provides intrinsic optical sections. Furthermore, compared to conventional microscopy, the light sheet method shows a reduction in photobleaching and a decrease in phototoxicity, and often enables much more scanning per sample. By rotating the specimen, the technique can image a virtually arbitrary 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 described by Ernst Abbe in 1873. A good approximation of the achievable resolution is the full width at half maximum (FWHM) of the point spread function, and a precision wide-field microscope with a high aperture and visible light typically reaches a resolution of about 250 nm. Super-resolution techniques allow images to be captured at a resolution higher than the diffraction limit. These can be broadly divided into two categories: "true" super-resolution techniques that capture information contained in evanescent waves, and "functional" super-resolution techniques that use experimental techniques and known limitations of the imaged material to reconstruct super-resolved images.

[0091] Laser microscopes use a laser illumination source in various forms of microscopes. For example, laser microscopy focused on biological applications uses ultrashort pulsed lasers, or femtosecond lasers, in several techniques including multi-photon fluorescence microscopy such as non-linear microscopy, saturation microscopy, and two-photon excitation microscopy (a fluorescence imaging method that enables imaging of biological tissues to a very high depth, e.g., up to 1 millimeter).

[0092] In an electron microscope (EM), a beam of electrons is used to irradiate a specimen and produce a magnified image. Electron microscopes have a higher resolution than light optical microscopes because they have a wavelength that is approximately 100,000 times shorter than that of visible light (photons). They can achieve a resolution better than 50 pm and a magnification of up to approximately 10,000,000 times, while a normal non-confocal light microscope is 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 an optical microscope that form a magnified image by focusing light on or through a specimen. Electron microscopes are used to observe a wide range of biological and inorganic specimens, including microorganisms, cells, large molecules, biopsy samples, metals, and crystals. Industrially, electron microscopes are often used for quality control and failure analysis. Examples of electron microscopy methods include transmission electron microscopy (TEM), scanning electron microscopy (SEM), reflection electron microscopy (REM) , scanning transmission electron microscopy (STEM), and low-voltage electron microscopy (LVEM).

[0093] A scanning probe microscope (SPM) is a branch of microscopes that forms an image of a surface using a physical probe that scans a specimen. 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 between the probe and the surface as a function of position. Examples of SPMs include atomic force microscope (ATM), ballistic electron emission microscope (BEEM), chemical force microscope (CFM), conductive atomic force microscope (C-AFM), electrochemical scanning tunneling microscope (ECSTM), electrostatic force microscope (EFM), fluid force microscope (FluidFM), force modulation microscope (FMM), function-oriented scanning probe microscope (FOSPM), Kelvin probe force microscope (KPFM), magnetic force microscope (MFM), magnetic resonance force microscope (MRFM), near-field scanning optical microscope (NSOM) (or SNOM, scanning near-field optical microscope, SNOM, piezoresponse force microscope (PFM), PSTM, photon scanning tunneling microscope (PSTM), PTMS, photothermal microscopy / spectroscopy (PTMS), SCM, scanning capacitance microscope (SCM), SECM, scanning electrochemical microscope (SECM), SGM, scanning gate microscope (SGM), SHPM, scanning Hall probe microscope (SHPM), SICM, scanning ion conductance microscope (SICM), SPSM spin-polarized scanning tunneling microscope (SPSM), SSRM, scanning spreading resistance microscope (SSRM), SThM, scanning thermal microscope (SThM), STM, scanning tunneling microscope (STM), STP, scanning tunneling potentiometry (STP), SVM, scanning voltage microscope (SVM), and synchrotron X-ray scanning tunneling microscope (SXSTM).

[0094] The unharmed tissue expansion microscope (exM) enables 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 prior to imaging, resulting in sub-diffraction limit structures within the size range visible by conventional diffraction-limited microscopes. ExM can image biological specimens at the voxel rate of diffraction-limited microscopes, but can image at the voxel size of super-resolution microscopes. The expanded sample is transparent and the expanded material is >99% water, so the water has a matching index. The techniques of expansion microscopy are known in the art as disclosed, for example, in Gao et al., Q&A: Expansion Microscopy, BMC Biol. 2017;15:50.

[0095] Screening method 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 intact tissue, the method comprising: (a) contacting an immobilized 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 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, the third complementary region of the first oligonucleotide being complementary to the third complementary region of the second oligonucleotide, the second complementary region of the second oligonucleotide being complementary to a second portion of the target nucleic acid, and 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 second oligonucleotide to generate a closed nucleic acid loop; and (c) performing rolling circle amplification in the presence of a nucleic acid molecule, using the second oligonucleotide as a template and the first oligonucleotide as a primer for a polymerase performing, including using to form one or more amplicons; (d) embedding one or more amplicons in the presence of a hydrogel subunit to form one or more hydrogel-embedded amplicons; (e) contacting one or more hydrogel-embedded amplicons having a barcode sequence with a pair of primers under conditions that permit ligation, wherein the pair of primers includes a third oligonucleotide and a fourth oligonucleotide, and ligation occurs only when both the third oligonucleotide and the fourth oligonucleotide ligate to the same amplicon; (f) repeating step (e); (g) imaging one or more hydrogel-embedded amplicons to determine in situ gene sequencing of a target nucleic acid in cells in a tissue sample; and (h) detecting a gene expression level of the target nucleic acid, wherein a change in the gene expression level of the target nucleic acid in the presence of 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 a tissue sample. Such screening methods include the steps of CISITS provided herein.

[0096] In some embodiments, detection includes performing flow cytometry, sequencing, probe binding and electrochemical detection, pH changes, catalysis induced by an enzyme bound to a DNA tag, quantum entanglement, Raman spectroscopy, terahertz wave technology, and / or scanning electron microscopy. In certain embodiments, flow cytometry is mass cytometry or fluorescence-activated flow cytometry. In some other embodiments, detection includes performing microscopy, scanning mass spectrometry, or other imaging techniques described herein. In such embodiments, detection includes determining a signal, e.g., a fluorescence signal.

[0097] As used interchangeably herein, "test agent", "candidate agent", and grammatical equivalents herein refer to any molecule being tested for activity in the subject assay (e.g., proteins (including proteins, polypeptides, and peptides herein), small molecules (i.e., having a size of 5 - 1000 Da, 100 - 750 Da, 200 - 500 Da, or less than 500 Da), or organic or inorganic molecules, polysaccharides, polynucleotides, etc.).

[0098] A variety of different candidate agents can be screened by the above method. Candidate agents include small organic compounds having a molecular weight greater 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, such as 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 can include functional groups necessary for structural interactions with proteins, such as hydrogen bonds, and can include at least an amine, carbonyl, hydroxyl, or carboxyl group, or at least two of the functional chemical groups. Candidate agents can include cyclic carbon or heterocyclic structures substituted with one or more of the above functional groups, and / or aromatic or polyaromatic structures. Candidate agents are also found in 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 This can be achieved. For example, numerous means are available for the random and directed synthesis of a wide variety of organic compounds and biomolecules, including the expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or can be readily generated. Additionally, libraries and compounds generated either naturally or synthetically can be readily modified through conventional chemical, physical, and biochemical means and used to generate combinatorial libraries. Known pharmacological agents can undergo direct or random chemical modifications such as acylation, alkylation, esterification, amidation, etc. to generate structural analogs. Furthermore, screening can be directed towards novel agents with unknown properties such as known pharmacologically active compounds and their chemical analogs, or those generated via rational drug design.

[0101] In one embodiment, the candidate modulator is a synthetic compound. Any number of means are available for the 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 hereby expressly incorporated by reference and discusses methods for generating novel compounds, including random chemical and enzymatic methods.

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

[0103] In one embodiment, the candidate agent includes proteins (antibodies, antibody fragments (i.e., fragments containing antigen-binding regions, single-chain antibodies, etc.), nucleic acids, and chemical moieties. In one embodiment, the candidate agent is a naturally occurring protein or a fragment of a naturally occurring protein. Thus, for example, cell extracts containing proteins, or randomly or directionally digested products of proteinaceous cell extracts may be tested. In this way, libraries of prokaryotic and eukaryotic proteins may be created 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, generally, as described in WO94 / 24314, the candidate agent is synthesized from a series of substrates that can be chemically modified. "Chemical modification" herein includes conventional chemical reactions and enzymatic reactions. These substrates generally include 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, tetracyclines, cephalosporins, and carbohydrates), steroids (including estrogens, androgens, cortisone, ecdyson, etc.), alkaloids (including ergot, vinca, curare, pyrrolizidine, and mitomycin), organometallic compounds, heteroatom-bearing compounds, amino acids, and nucleosides, but are not limited thereto. Chemical (including enzymatic) reactions may be carried out in part to form new substrates or candidate agents, which can then be tested using the present invention.

[0105] Devices and Systems Also included are devices for implementing aspects of the CISITS method. The subject devices may include, for example, sample chambers, imaging chambers, electrophoresis apparatuses, flow chambers, microscopes, needles, tubes, and pumps. ​

[0106] The present disclosure also provides a system for implementing the subject method. The system can include one or more of the modules described herein, such as a power source, a refrigeration unit, a waste unit, a heating unit, a pump, and the like. The system can also include any of the reagents described herein, such as an imaging buffer, a wash buffer, a stripping buffer, Nissl and DAPI solutions. The system according to certain embodiments can also include a microscope and / or a related imaging device, 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, and the like.

[0107] An exemplary fluid system for implementing the CISITS method described in this specification is shown in FIG. 7. The system described in this specification includes a fluid device having an unpressurized sample chamber with a lid having an interface tube, wherein a first end of the interface tube is held by the lid and a second end of the interface tube is positioned over a 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 such that as long as liquid remains in the sample chamber, the liquid in the sample chamber remains in contact with the bottom of the interface tube. Sufficient space is left between the bottom of the sample interface tube and the sample chamber such that the 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 arranged at a polar angle with respect to the tissue sample plane. A fluid line is connected to the first end of the interface tube such that fluid flows through the fluid line into the interface tube and out onto the tissue sample in the sample chamber from the second end of the interface tube. To maintain the sample chamber at cryogenic temperatures, a cryostat may be included in the fluid system. The fluid system also includes an imaging system, a pump, and a processor unit configured to implement the CISITS method described in this specification.

[0108] In some embodiments, the system enables the automation of CISITS, which is the process described herein, and which includes, but is not limited to, iterative rounds of probe hybridization with DNA embedded in a gel, ligation of fluorescently labeled oligonucleotides onto these probes, washing away of excess probes, imaging, and cleavage of fluorophores from the probes prior to the next round of sequencing. In some embodiments, the system may enable continuous operation. In some embodiments, the system includes an imaging chamber for flowing sequencing chemistries involved in in situ DNA sequencing of a sample. In some embodiments, a system of fluids and pumps controls the delivery of sequencing chemistries to the sample.

[0109] The buffer can be added / removed / recycled / exchanged by means of one or more ports and, optionally, the use of tubes removably or permanently attached to one or more components of the device, such as tubes, pumps, valves, or any other suitable fluid handling and / or fluid manipulation device. For example, a first tube having first and second ends can be attached to a first port, a second tube having first and second ends can be attached to a second port, the first end of the first tube is attached to the first port, the second end of the first tube is operably connected to a container, such as a cooling unit, heating unit, filtration unit, waste container, etc., the first end of the second tube is attached to the second port, and the second end of the second tube is operably connected to a container, such as a cooling unit, beaker on ice, filtration unit, waste container, etc.

[0110] In some embodiments, the multi-directional selector valve is interfaced with a pump. The multi-directional selector valve is used to switch the currently open fluid line to enable various operating modes. The pump can be used to draw in or push out a certain 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 such that the pump can draw the selected reagent or buffer into the system. The fluid system may further include an adjustable stage that supports the reagent tray and a position sensor that enables positioning of the adjustable stage to allow selection of a desired reagent from the reagent tray fluidly connected to the multi-directional selector valve. A cooler may be included in the fluid system to maintain the desired temperature of the reagent. The pump can also be used to draw reagent from a sample well through an interface tube. Additionally, the pump can be used to push a volume through an interface tube, for example, into a sample well or into a waste container. Alternatively, the pump can push the volume back in reverse into a reagent bank (which can be used for purification purposes).

[0111] When practicing the CISITS method, it is preferred to use fresh reagents, particularly for SEDAL sequencing, rolling circle amplification, and ligation. In some embodiments, a disposable reagent tray containing reagents for practicing the CISITS method is used in the fluid system to enable the use of fresh reagents that are convenient for the user.

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

[0113] In some embodiments, the present system is surrounded by a container that blocks ambient light. The container can include several doors and drawers for accessing various internal components of the system. For example, the system may include a drawer for inserting a sample into the system or removing it, and a drawer for inserting a reagent into the system or removing it. Other components of the enclosure can provide access for service professionals to change, upgrade, or repair the internal parts of the system. The enclosure can also be acoustically attenuating.

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

[0115] Usefulness The devices, methods, and systems herein find several uses in the art such as biomedical research and / or clinical diagnostics. For example, in biomedical research, the uses include spatially resolved for basic biology or drug screening It includes, but is not limited to, gene expression analysis. In clinical diagnosis, the uses include, but are not limited to, the detection of gene markers such as diseases, immune responses, bacterial or viral DNA / RNA in patient samples. Examples of the advantages of the methods described herein include efficiency, taking only 3 or 4 days to obtain final data from a biological sample, providing a much faster rate than existing microarray or sequencing technologies, being highly multiplexed (up to 1000 genes), having 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 embodiments, CISITS can be applied to the study of molecularly defined cell types and activity-regulated gene expression in the mouse visual cortex, can be extended to larger 3D tissue blocks, and can visualize short-range and long-range spatial organization of cortical neurons at volume scales that were 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 presents special challenges that are well-suited for CISITS analysis. For example, polymorphic activity-regulated gene (ARG) expression observed across different cell types is likely to depend on both intrinsic cell biological properties (e.g., signaling pathway component expression), and extrinsic properties such as the neural circuit anatomy that sends external sensory information to different cells (here, the visual cortex). In such cases, in situ transcriptomics exemplified by CISITS can effectively link imaging-based molecular information with anatomical and activity information, and thus elucidate brain function and dysfunction.

[0118] The devices, methods, and systems disclosed herein enable the removal of cellular components, such as lipids, that normally provide structural support but impede visualization of intracellular proteins and molecules, while maintaining the three-dimensional architecture of cells and tissues because the sample is cross-linked to a hydrogel that physically supports the ultrastructure of the tissue. This removal renders the interior of the biological specimen substantially transparent to light and / or macromolecules, enabling microscopic visualization of the interior of the specimen, such as cells and subcellular structures, without the time-consuming and destructive sectioning of the tissue. Typically, the purification and permeabilization, which are typically performed in separate steps, can be combined in a single step to remove cellular components, so this procedure is also faster than procedures commonly used in the art. Additionally, the specimen can be repeatedly stained, unstained, and restained with other reagents for comprehensive analysis. Further functionalization at the polymerizable acrylamide sites enables the covalent fixation of amplicons 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 a 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 a subject affected by a disease or disorder (e.g., cancer status, cancer stage, identification of 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, e.g., no response at all to allogeneic hematopoietic stem cell transplantation, chemotherapy, radiation therapy, antibody therapy, small molecule compound therapy), and use of a treatment method (e.g., monitoring a subject's condition to provide information regarding the effect or efficacy of the therapy). For example, a biopsy can be prepared from cancerous tissue and microscopically analyzed to determine the type of cancer, the extent to which the cancer has developed, whether the cancer responds to therapeutic intervention, etc.

[0120] The subject devices, methods, and systems also their effects on tissues or diseases Provided are useful techniques for screening candidate therapeutic agents. For example, a subject, such as a mouse, rat, dog, primate, human, etc., can be contacted with a candidate agent, its organs or biopsies can be prepared by the method of the subject, and the prepared specimens can be microscopically analyzed for one or more cell or tissue parameters. A parameter is a quantifiable component of a cell or tissue, and in particular, is a component that can be accurately measured, preferably in a high-throughput system. Parameters can be any cell component or cell product that includes cell surface determinants, receptors, proteins or their conformation or post-translational modifications, lipids, carbohydrates, organic or inorganic molecules, nucleic acids, such as mRNA, DNA, etc., or moieties derived from such cell components or combinations thereof. Most parameters provide a quantitative readout, although in some cases, semi-quantitative or qualitative results are acceptable. The readout may include a single determined value or may include, for example, an average, median or variance. Characteristically, the range of readout values for a parameter is obtained for each parameter from the multiplicity of the same assay. Variability is expected, and the range of values for each set of test parameters is obtained using standard statistical methods using 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, the effect of changing disease progression, etc. In some embodiments, the screen includes comparing the analyzed parameters to those from a control or reference sample, such as a sample prepared similarly from a subject not contacted with the candidate agent. Candidate agents of interest for screening include a number of chemical classes, mainly organic molecules, including known and unknown compounds that can include organometallic molecules, inorganic molecules, gene sequences, etc. Candidate agents 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 to evaluate candidate drugs, including toxicity tests and the like.The evaluation of tissue samples using the subject methods can include, for example, genetic, transcriptomic, genomic, proteomic, and / or metabolomic analyses.

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

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

[0123] Examples of non-limiting aspects of the present disclosure The above-described aspects (including embodiments) of the present subject matter can be beneficial alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing description, specific non-limiting aspects of the present disclosure numbered 1 - 47 are provided below. As will be apparent to those skilled in the art upon reading the present disclosure, each of the individually numbered aspects can be used or combined with any of the foregoing aspects or any of the aspects following the individually numbered aspects. This is the It is intended to provide support for all such combinations and is not limited to combinations of the modes explicitly provided below.

[0124] 1. A method for in situ gene sequencing of a target nucleic acid in cells in intact tissue, the method comprising: (a) contacting an immobilized and permeabilized intact tissue sample with at least a pair of oligonucleotides under conditions that allow specific hybridization, wherein the pair of oligonucleotides comprises a first oligonucleotide and a second oligonucleotide, each of the first oligonucleotide and the second oligonucleotide comprises a first complementary region, a second complementary region, and a third complementary region, and the second oligonucleotide further comprises a barcode sequence, 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, the second complementary region of the second oligonucleotide is complementary to a second portion of the target nucleic acid, and 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 generate a closed nucleic acid ring; (c) pre-incubating the tissue sample with a DNA polymerase for a sufficient time 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 such that one or more amplicons are generated, wherein the ligated second oligonucleotide functions as a template and the first oligonucleotide functions as a primer for DNA polymerase, (e) Embedding the tissue sample in a hydrogel either before or after any one of steps (a) to (d), (f) Crosslinking one or more amplicons to the hydrogel, (g) Purifying the hydrogel to enhance its transparency, (h) Contacting one or more hydrogel-embedded amplicons having barcode sequences with a read primer and a fluorescently labeled probe, wherein the probe comprises an oligonucleotide comprising a first thiol group covalently bound to a fluorophore comprising a second thiol group by a disulfide bond between the first thiol group and the second thiol group, (i) Ligating the read primer and the fluorescently labeled probe, wherein ligation occurs only if both the read primer and the fluorescently labeled probe are complementary to adjacent sequences of the same amplicon, (j) Contacting the hydrogel with an anti-fading buffer containing an antioxidant, (k) Imaging one or more hydrogel-embedded amplicons to determine the positioning of the target nucleic acid in cells in intact tissue undergoing in situ gene sequencing, wherein imaging is performed in the presence of the anti-fading buffer, (l) Contacting the hydrogel with a reducing agent to effect reduction of the disulfide bond and cleavage of the fluorophore from the probe, (m) Removing the fluorophore from the hydrogel, (n) Repeating steps (h) to (m), A method comprising.

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

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

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

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

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

[0130] 26. The method according to any one of aspects 1 to 25, wherein purification comprises substantially removing a plurality of cell components from the hydrogel-embedded tissue sample. 27. The method according to aspect 25 or 26, wherein purification comprises substantially removing lipids and proteins from the hydrogel-embedded tissue sample. 28. The method according to any one of aspects 0 to 27, wherein imaging comprises imaging one or more hydrogel-embedded amplicons using confocal microscopy, two-photon microscopy, bright-field microscopy, label-free tissue expansion microscopy, and / or CLARITY™ optimized light-sheet microscopy (COLM). 29. The method according to any one of aspects 1 to 28, wherein the label-free tissue has a thickness of 5 to 20 μm. 30. The method according to any one of aspects 1 to 28, wherein the label-free tissue has a thickness of 50 to 200 μm.

[0131] 31. The method according to any one of aspects 1 to 30, further comprising slicing the label-free tissue and placing the slice of the label-free tissue flat on a surface. 32. The method according to aspect 31, wherein a metal ring is pushed down onto the slice and used to transfer the slice flat on the surface.

[0132] 33. A fluid system comprising: a) a device comprising an unpressurized sample chamber; b) a lid that covers the top of the sample chamber; c) an interface tube, wherein a first end of the interface tube is held by the lid and a second end of the interface tube is positioned over a tissue sample in the sample chamber in sufficient proximity to the bottom edge of the sample chamber such that, as long as liquid remains in the sample chamber, the liquid in the sample chamber remains in contact with the bottom of the interface tube; 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 onto the tissue sample in the sample chamber from the second end of the interface tube; 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 fluid system according to aspect 33, wherein the sample chamber is a well of a multi-well plate or a slide chamber. 35. The fluid system according to aspect 33 or 34, wherein the interface tube is arranged at a polar angle with respect to the tissue sample plane. 36. The fluid system according to any one of aspects 33 to 35, further comprising a cryostat for maintaining the sample chamber at a cryogenic temperature. 37. The fluid system according to any one of aspects 33 to 36, further comprising a multi-directional selector valve interfaced with a pump. 38. The fluid system according to aspect 37, further comprising a reagent tray comprising one or more containers or wells containing reagents for performing in situ gene sequencing of target nucleic acids in cells in intact tissue, the reagent being fluidly connected to the multi-directional selector valve.

[0134] 39. The fluid system according to aspect 38, further comprising an adjustable stage for supporting the reagent tray, a cooler, and a position sensor enabling movement of the adjustable stage to enable selection of reagents from the reagent tray. 18. The fluid system according to aspect 38. 40. The fluid system according to any one of aspects 37 to 39, further comprising a buffer tray comprising one or more containers or wells containing buffer, the buffer being fluidly connected to the multi-directional selector valve. 41. The fluid system according to any one of aspects 38 to 40, wherein the reagent tray and / or the buffer tray is disposable. 42. The fluid system according to any one of aspects 37 to 41, further comprising a waste container fluidly connected to the multi-directional selector valve. 43. The fluid system according to any one of aspects 33 to 42, wherein the pump is a syringe pump.

[0135] 44. The fluid system according to any one of aspects 33 to 43, wherein the imaging system includes a microscope that performs confocal microscopy, spinning disk confocal microscopy, light sheet microscopy, lattice light sheet microscopy, or wide field microscopy. 45. The fluid system according to any one of aspects 33 to 44, further comprising a container surrounding the fluid system, wherein the container blocks ambient light. 46. The fluid system according to aspect 45, wherein the container surrounding the fluid system includes one or more doors or drawers. 47. The fluid system according to aspect 45 or 46, wherein the container surrounding the fluid system is an acoustic attenuation container. 48. The fluid system according to any one of aspects 33 to 47, further comprising means for cryosectioning a tissue sample.

Examples

[0136] The following examples are presented to provide those skilled 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 that the inventors regard as the invention, nor are they intended to represent that the following experiments are all or the only experiments to be performed. Although efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), some experimental error and deviation should be considered. Unless otherwise indicated, parts are by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric or near atmospheric pressure.

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

[0138] The present invention is described with respect to specific embodiments found or provided by the inventors so as to include preferred modes for carrying out the present invention. It should be understood by those skilled in the art that, in light of the present disclosure, numerous modifications and changes can be made to the specific embodiments illustrated without departing from the intended scope of the invention. For example, due to codon redundancy, changes to the underlying DNA sequence can be made without affecting the protein sequence. By considering biological functional equivalence, changes to the protein structure can be made without affecting the biological action in terms of type 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 and bound to a hydrogel. The first publication of STARmap demonstrated the use of combinatorial sequencing at a sample size of 0.0034 cubic millimeters and the use of sequential sequencing at a sample size of 0.238 cubic millimeters. Considerable human labor was required to collect these data, and even just the readout stage of sequencing required several days of continuous labor for a single sample. The entire adult mouse brain is approximately 400 cubic millimeters, i.e., more than 110,000 times the volume of the previously demonstrated combinatorial sequencing sample. As intact tissue sequencing approaches the scale for increasing volume and throughput, it is very important to maximize their robustness (to prevent the compound accumulation of errors) and enhance parallelism and throughput. This is particularly true when these techniques transition from the laboratory to clinical and industrial workflows.

[0140] The new method described in this specification targets several different robustness and throughput aspects of the previous STARmap technique, including both sequencing chemistry and automated execution of sequencing. This new method is called "Clinical and Industrial Scale In Situ Tissue Sequencing (CISITS)". The chemical aspect of the new method focuses on increasing the signal-to-noise ratio (SNR) of labeled molecules, eliminating carry-over signals across rounds, achieving reliable efficiency and chemical success across the protocol, and enabling combinatorial sequencing in thick tissues. The new automated method includes a combination of a fluid system where the sample chamber is no longer pressurized, a system integration method for high system robustness, and a method for parallelizing across samples. The CISITS software control method manages the automated fluid system and coordinates a robust fluid exchange process while maintaining the sequencing workflow in the event of a failure. The integration of CISITS with microscope hardware provides system integration that simplifies, accelerates user interaction, and enables large-scale field acquisition to enhance throughput.

[0141] CISITS (1) New method for high sequencing SNR In the original version of STARmap, during sequencing, amplicons are labeled by binding fluorescently labeled oligos to the appropriate amplicons such that they remain bound at room temperature after washing. For both post-ligation washing and imaging, a “wash and image” buffer is used, which is formulated with 2× SSC, 10% formamide, and water. Imaging in this buffer suffered from photobleaching of the fluorophore and loss of signal with longer exposure times. This was a problem both in the initial rounds of sequencing by wide-field image tiling (when signals were highest), which increased the total amount of light delivered to the edges of the field of view and decreased the SNR and detectability of individual amplicons. This was particularly problematic in later rounds of sequencing, which undergo a characteristic decrease in SNR due to the nature of the SEDAL sequencing chemistry. In later rounds, especially for fluorophores with longer wavelengths, a complete barcode read is required to successfully sequence, and poor SNR begins to impair the ability to extract high-quality reads from the molecules, decreasing the overall yield of sequenced molecules.

[0142] The wash and image buffer formulation was changed to include antioxidant compounds such as N-propyl nitrate. For example, the new composition of the wash and image buffer is 2× SSC, water, and 10% of 20% N-propyl gallate dissolved in formamide, with a final concentration of 10% formamide and 2% N-propyl gallate. Inclusion of an antioxidant compound in the wash and image buffer functions as an anti-bleaching agent for the fluorophore. This reduces photobleaching during tiling, significantly enhances the SNR of sensitive fluorophores, and enables higher SNR imaging of thicker samples. In the case of a fixed exposure time with an antioxidant compound, the SNR is increased by increasing the concentration of unbleached fluorophore per dot over the exposure. Inclusion of the antioxidant also removes and reduces the return effect of longer exposure times (caused by the limited fluorophore lifetime before bleaching), allowing for a linear increase in SNR by increasing the exposure time as needed.

[0143] (2) Improvement in signal separation from round to round In the continuous readout approach for STARmap array determination, barcode - amplified amplicons from individual genes are probed only in a single round such that a given color channel conveys information about different genes from round to round. This continuous readout approach is used in thicker tissue sections where it is more difficult to control the size of the amplicons and maintain separation of the amplicons across rounds, or in samples where the number of genes to be read out is less and emphasis is placed on volume throughput, which is achieved by using a lower magnification objective lens and camera binning, e.g., 4×4 binning of pixels per image. The success of this continuous approach requires that signals from a given color channel be completely eliminated from round to round. Otherwise, for example, remaining signals from highly expressed genes in one round may be misidentified as signals from low - expressed genes in the next round.

[0144] This was solved by increasing the concentration of formamide from 60% to 80% and increasing the length and number of formamide stripping cycles in all rounds. However, this improved the efficiency of signal removal for all genes and completely removed the signals. Its efficiency is subject to room - temperature changes and may add a significant additional time per sample. Additional formamide exposure may degrade the quality of the sample over time and across sequencing rounds.

[0145] To more completely eliminate round-to-round signals, another aspect of CISITS involves SEDAL-based sequencing readout combined with fluorophore cleavage (as opposed to probe cleavage). Incorporating a thiol bond between the fluorophore and the rest of the oligo enables cleavage of the fluorophore in a reducing environment such as a TCEP solution, which is used in other in situ sequencing approaches. However, in the SEDAL sequencing of STARmap, high SNR labeling of amplicons occurs during the ligation reaction, and the ligation buffer contains the reducing agent DTT, which has been found to dramatically reduce signals from thiol-containing fluorescent oligos.

[0146] CISITS re-prepares the SEDAL sequencing / ligation buffer to remove the DTT component. It was found that there is little difference in specificity or SNR from non-thiol-containing fluorescent oligos, and thiol-containing fluorescent fluorophore-labeled oligos can be bound to target amplicons. Removal of DTT additionally allows thiol-containing fluorophore probes to remain stable at 4°C in the ligation mixture for at least 24 hours, with minimal subsequent loss of labeling efficacy, facilitating automation. After imaging of the sequencing rounds, addition of a buffer containing the reducing agent TCEP for 30 minutes completely removes the punctate fluorescence signals, and subsequent short PBSTw washes remove the diffusive fluorescence signals.

[0147] This aspect of the new method has the advantages of robust signal removal, rapid round times due to reduced signal removal / wash times, and in particular conveys a certain tissue robustness, enabling more total rounds without compromising sample integrity. Using SEDAL sequencing with thiol-based chemical cleavage in CISITS combines the advantages of SEDAL sequencing in terms of specificity and SNR with the robustness and rapidity of the round-to-round cycles from fluorophore cleavage.

[0148] (3) New Methods for Robustness in the CISITS Protocol Before and During Array Determination Tissue Section Collection For intact tissue combinatorial array determination, fresh frozen tissue sections are collected by cryosectioning and placing the sections on fresh silane-coated wells or slides. To facilitate high-throughput, parallel processing of samples, it is preferred to collect samples in multi-well plates such as 24-well plates. However, it is difficult to transfer frozen sections to the bottom of a silane-coated plate without tissue disruption, folding, or tearing, which can interfere with subsequent data collection and analysis. Furthermore, tissue collection and preparation prior to array readout need to be highly parallelized across samples, and optimal sample collection is essential. The STARmap method does not detail a robust sample collection process, and standard approaches have a very high failure rate per section, resulting in the loss of many sections and inefficient use of space on the plate.

[0149] As part of CISITS, a new method was devised to keep the sample collection wells cold in a cryostat with small thin-walled metal rings of 8 or 10 mm diameter to ensure that frozen cross-sections are placed flat on the surface for downstream processing. After the sections are collected, the metal ring is pushed down onto the section so that the edge of the ring contacts the frozen cutting medium evenly surrounding the section. The ring and flat section can then be picked up using forceps and transferred to the appropriate surface, which is warmed on the underside with a finger just prior to transfer. The metal ring facilitates accurate flat placement of the section on the silane surface, and the warmth from the finger separates the section from the ring and attaches it to the well. This new method significantly guarantees section integrity and throughput.

[0150] CISITS Formulation of NHS Ester Buffer of Acrylic Acid Previously formulated AA NHS ester buffers in the STARmap method were not pH buffered. In the new CISITS method containing 20 mM MOPS pH 7.7 in the AA NHS ester buffer, the pH buffers the solution at a more optimal pH for the AA NHS ester, making the subsequent NHS ester reaction reliable and efficient.

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

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

[0153] The ligation mixture for the SEDAL sequencing rounds can be premixed and stored at 4°C for at least 24 hours. The signal is optimal when the SEDAL ligation round mixture is freshly made every 24 hours of sequencing, provided it is stored at 4°C. During automated sequencing, additional SEDAL reagents for rounds >4 may be freshly added to the sequencing device after several rounds of operation are completed.

[0154] TCEP buffer is optimally used when freshly made prior to each sequencing run, but maintains efficiency over the sequencing cycle.

[0155] (4) New method for combinatorial labeling of nucleic acids in thicker tissues The main limitation to the throughput of the STARmap method is combinatorial It is impossible to perform sequence determination. This limitation stems from several causes. First, since 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 subsequent efficiency can be very limited. An additional limitation is the uniformity in the control of the size of RCA amplicons. Since the diffusion characteristics of thicker tissues are difficult, under the conventional STARmap method, it is not possible to obtain uniformly sized amplicons suitable for multiple rounds. Furthermore, the cost of obtaining probes suitably modified for direct incorporation into hydrogels (before ligation and amplification) can be high in the case of a large number of genes.

[0156] In the new method, several approaches have been devised to break through these limitations in order to enable large-scale sequencing of tissues. These new methods provide several different means for obtaining thick-section combinations and high-throughput sequencing.

[0157] In the first example of this new CITSIT method, as in the STARmap method, probes containing appropriate end modifications such as acridite are used for hybridization. They 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, a new method is used to perform amplification to achieve sufficiently uniform ligation and amplification efficiency across the sample. First, an RCA mixture is formulated to contain the enzyme and buffer but exclude dNTPs, thereby preventing any amplification from proceeding. This mixture is added to the ligated sample and allowed to diffuse into the sample until it becomes uniform, for example, over a period of 2 days. By removing the dNTPs, the slow enzyme diffusion process can proceed to equilibrium. After adding the dNTP-free RCA mixture and allowing it to equilibrate, an RCA mixture with an excess concentration of dNTPs is added to the sample. Since dNTPs are small molecules and have a high concentration gradient outside the sample, they rapidly equilibrate into 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 the optimal temperature for amplification and amplification is allowed to proceed for several hours, producing uniform and appropriately sized amplicons for subsequent rounds of combinatorial sequencing.

[0158] In a second example of the new method, the endogenous RNA is first bound to the hydrogel prior to hybridization. This can be individually replaced via a binding chemistry such as label X, or by binding to the 5’ methyl RNA mRNA cap, or by very stable hybridization to the polyA tail, or by subsequent target hybridization, but covalently either by short condensation probes that serve to hold the endogenous RNA in the hydrogel by collectively having sufficient long-term stability or by compounds that polymerize in the hydrogel mediating interaction with the endogenous RNA. After formation of the hydrogel embedding the RNA, the sample may be actively digested and permeabilized such that a clear hydrogel containing mostly endogenous RNA is obtained. This resulting hydrogel has superior diffusion properties compared to non-digested tissue. Probe hybridization, ligation, and RCA can then be carried out with appropriate incubation times, but the method for uniform RCA amplification described in the first example ensures compatibility with downstream combinatorial sequencing. Following RCA, the amplicons are ligated to the hydrogel according to the approach described above with the thin-section STARmap method for long-term stability prior to proceeding to combinatorial multiple-round sequencing.

[0159] Since the thickness of these samples is increased by orders of magnitude compared to the thin-section method, incorporating propyl gallate or other antioxidant compounds in the wash and imaging buffers is essential to prevent fading when the large depth of the sample in Z is imaged.

[0160] New Automated Fluid System Hardware Method for CISITS Conventional automated methods for STARmap array determination consisted of a fluid system where the array determination reagent was pushed across the sample in a pressurized, airless flow cell and drawn in. For the accurate and robust functioning of this flow cell-based system, it was necessary to remove air bubbles from the fluid lines, appropriately manage the flow of the reagent in the flow cell so that the reagent was completely exchanged at each step of the array determination, properly prime the system so that the correct amount of each reagent was filled, and properly seal the flow cell when connecting to the fluid. Failure to meet any of these requirements at any point during multi-day array determination could lead to array determination failure. Meeting these requirements was complicated by the heterogeneous shapes and sizes of the samples on the flow cell. Additionally, there was a fundamental non-exchangeability between the STARmap method for sample preparation prior to array determination and the aforementioned method for automated STARmap array determination in the flow cell. That is, existing STARmap methods enable the parallel preparation of many samples adhered to individual wells of a multi-well plate, but do not specify any method for transferring hybridized, ligated, amplified, and gelled samples to the flow cell, which is difficult due to the adhesive properties of the samples to the bottom of the sample preparation plate. A method for performing the STARmap sample preparation method directly on the flow cell from the beginning has not been described, which, in addition to the loss of ease of parallelization across individual samples, suffers from evaporation and liquid management problems. Finally, previous STARmap automated methods described the determination of only a single sample (or flow cell) at a time, severely limiting the throughput of the method. A new method has been developed to address many of these robustness and throughput problems of previous methods.

[0161] CISITS fluid system New method for sample interface connection The CISITS fluid system adds and removes sequencing reagents from a sample. Unlike previous methods, this new method uses an unpressurized sample chamber that can accommodate air above the sample. By choosing to simply immerse the sample in the reagent to relax the requirements for the flow of the reagent to the sample, the robustness and simplicity of the system are significantly improved, while at the same time significantly promoting the parallelization of automated sequencing across different sample chambers.

[0162] In this system, one or more samples are positioned in the sample chamber, which is the glass-bottom well of a multi-well plate (for optimal compatibility with the pre-sequencing CISITS method), or any glass-bottom chamber where the air in the chamber can exchange with room air and the flow pressure can be relaxed. In one example, samples are prepared using the CISITS method in the wells of a 6-, 12-, or 24-well multi-well glass-bottom plate. The fluid system is coupled to the sample via a custom well plate lid that holds a rigid fluid tube (such as a metal tube) immediately above the sample near the bottom edge of the sample well (interface tube). The fluid line is coupled to this tube such that fluid passes through the fluid line, into the interface tube, and immerses it over the sample in the well. To remove fluid from the sample, the fluid is suctioned out of the sample well through the interface tube. Different custom lids for the CISITS fluid system are designed to accommodate different sample chamber formats, including multiple arrangements of various different types of multi-well plates or slide chambers. In the case of a multi-well plate, the system can couple to multiple different sample wells simultaneously, provide access to different sample wells (to be sequenced sequentially), or alternatively provide parallel sequencing capabilities across the wells (described below).

[0163] The custom CISITS lid that couples the fluid system to the sample can be easily placed on top of or removed at any time to allow air exchange to the sample chamber or plate It is not sealed on the lid. However, the fluid interface tube is accurately positioned in the lid such that as long as liquid remains in the chamber, the liquid in the sample chamber remains in contact with the bottom of the tube (through agglomeration and adhesion). This ensures that all or almost all of the reagent is removed from the sample well in each reagent removal step, guaranteeing a robust automated process. Sufficient space is left between the bottom of the sample interface tube and the sample chamber, and the liquid added to the sample does not pressurize the interface tube; instead, it spreads throughout and immerses the sample. The sample interface tube can be adapted to a container with many tubes having many different sample chambers and can be arranged at a polar angle with respect to the plane of the sample such that each chamber has an interface tube. Alternatively, a custom CISITS lid can allow the user to place one or more interface tubes in several desired sample chambers such that the tubes are reliably guided to the exact position that provides a robust fluid exchange. These custom lids may be machined or 3D printed to specification and may be consumable or reusable.

[0164] New method for continuous fluid flow through reagent selection, sample, and discard The fluid system for CISITS is centered around a syringe pump and draws in a desired amount of reagent into the pump through a series of selector valves. In the case of single sample operation, the pump in this new method has several operating modes. It can draw in reagent from a multi-port selector valve coupled to a reagent reservoir or draw in reagent from a sample well through an interface tube. Once the pump has drawn in some volume, it can either extrude the volume through the interface tube into the sample well, extrude the volume into a waste container, or extrude the volume back into the reagent reservoir (which can be used for cleaning purposes). These different operating modes are mediated by a multi-directional selector valve (e.g., 3-way) interfaced with the pump that switches which fluid lines are currently open. Thus, an example of the normal mode of operation for continuous sequencing is to open only the pump selector to the reagent selector line, open the desired multi-port selector lines for the reagent of interest, draw the selected reagent from the reagent reservoir through the multi-port selector into the pump, allow equilibration to occur to control the various flow times due to different liquid properties, open the pump selector only towards the sample well, raise the syringe pump, extrude the reagent through the sample fluid line and interface tube to immerse the sample, draw a small amount of air into the pump instead of reagent and extrude it into the sample, wash out any remaining reagent in the sample fluid line, repeat this process to completely 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 the liquid from the sample into the pump through the interface tube to allow equilibration, open only the pump selector port to waste, raise the pump and extrude the contents into waste, and repeat this liquid removal process for a sufficient time to completely remove the desired amount from the sample. This can be considered the reagent addition, incubation, and removal cycle in the normal operation of the CISITS method. The cycle is repeated for either the same reagent or a different reagent as the sample progresses through the CISITS sequencing process.This cycle process of the fluid system, unlike the flow cell process, leaves the sample without liquid for a short time. However, since the CISITS method uses tissue embedded in a hydrogel, the sample does not dry out on this time scale and thus does not always need to be immersed. Additionally, since the sample adheres to the bottom of the chamber and is imaged with an inverted microscope, air cannot move between the bottom of the well and the sample, so air bubbles are not a problem with this method.

[0165] This non-pressurized sample chamber method for the fluid system enables various robustness methods such as the aforementioned air movement through the system after reagent addition, ensuring complete addition of the reagent to the sample. Additionally, it enables a larger volume of reagent to be drawn out by the pump than would otherwise be possible, introducing an additional air volume to the pump, while ensuring that any limited quantity of reagent is completely drawn out of the reagent container. The fluid system of this method can bypass the sample by drawing liquid into the pump and pushing it directly into the waste container, facilitating the washing process by drawing water, diluted bleach, or other cleaning fluid into the pump, purifying it, and pushing it out directly for disposal. Alternatively, water or other cleaning fluid can be pushed into the reagent container as needed, for example, to clean the reagent fluid line.

[0166] New method of parallel fluid flow This new CISITS method, with the sample chamber not pressurized, greatly facilitates the parallelization of 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 individually (enabling addressable access to different sample wells) or multiple can be opened simultaneously, allowing fluid to be delivered in parallel by the pump to multiple samples. By coupling the output fluid lines from this sample selector valve to each different sample interface tube and sample, parallel array determination using CISITS can be achieved, providing a substantial throughput in the array determination of multiple samples. For example, instead of a series of array determination processes that take about 48 hours each to determine one sample at a time, multiple samples can be determined simultaneously or alternately, such that the imaging acquisition system becomes the limiting process instead of the array determination cycle or incubation time. For example, in the case of a single array determination 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 results in a four-fold throughput speedup (it takes one-fold the time to image all five samples in each round), or alternatively, operating mutually or alternately, imaging acquisition is almost always performed following the first round, resulting in an almost five-fold speedup (less than the time for one alternating round). Thus, this new method results in an almost linear increase in the throughput of the array determination of intact tissue, with the number of parallel array determined samples up to the number of samples where the imaging acquisition system becomes limiting.

[0167] New Method for Reagent Management System In previous STARmap automated alignment methods, reagents remained cooled via different sample tubes either within a cooling block or within tubes at room temperature. Each reagent was directly coupled to the fluid system, and the user had to switch reagents by directly coupling and decoupling the reagent from the fluid system during execution.

[0168] In the new method, a reagent management system abstracts this process for the user, for example, simply by easily providing a disposable reagent tray that can be easily placed within the fluid device. The device places the appropriate portion of this reagent tray onto an internal cooling block and automatically couples the fluid line to the reagent. The reagent containers or wells within the reagent tray have sufficient heat transfer characteristics to enable coupling with a cooler unit. After completion of the CISITS execution / automated fluid operation, the tray can be discarded and a new tray used for a new execution. The user fills the appropriately labeled area of the reagent tray with the appropriate reagent and then places it within the liquid system. The reagent tray can vary the volume of different reagents depending on the throughput of the system and whether samples are being aligned by the CISITS method in parallel or sequentially.

[0169] To ensure the freshness of the alignment reagents, the tray may optionally be partially removed during the execution of the alignment to place the next few rounds of fresh mixed SEDAL reagents.

[0170] This new method for reagent management simplifies and speeds up the initialization of CISITS execution and provides a robust process that prevents the user from interrupting the normal operation of the system fluid.

[0171] New method for enclosing a CISITS alignment system The entire array determination system is surrounded by a container that blocks light while providing appropriate doors and drawers for accessing the various internal components of the system. Some of the drawers are intended for the normal use of the system, such as drawers for inserting a sample into the system or removing it, and drawers for inserting reagents into the system or removing them. Other components of the enclosure provide easier access for service professionals to change, upgrade, or repair the internal parts of the system. The enclosure may include acoustic attenuation.

[0172] New method for robust operation of fluid systems The CISITS automated array determination system includes a plurality of methods for ensuring the robustness of automated operation and a low failure rate. These methods include, for example, detectors of fluid flow through sample fluid lines to ensure fluid delivery, imaging objectives and absorbent materials surrounding electrodes to detect the outflow of reagents from sample containers, whose activity can stop array determination to prevent catastrophic damage, confirmation that the cooler has reached the appropriate temperature before filling with reagents, confirmation to detect proper filling of the CISITS reagent / tray system, periodic system cleaning automatically performed at the end of each array determination to prevent clogging of fluid lines due to the accumulation of salts or other substances, and intermittent cleaning performed on the system prior to execution if the system has not been recently used. In the cleaning automatically performed at the end of array determination, a dilute bleach or other cleaning solution is moved into the pump, then discarded, followed by multiple rounds of washing with water to completely remove the bleach or cleaning solution. After the automated procedure is complete, the system requests that the user place cleaning sample containers in a format consistent with that previously used by the user so that the bleach or cleaning solution and water can pass through the sample fluid lines and tubes without damaging the arrayed samples, or that this cleaning can be performed automatically on the sample containers with the user's consent. Finally, air passes through the system to remove any liquid remaining in the fluid lines. Intermediate cleaning can be initiated by the user using a cleaning sample plate during the execution of array determination, provided that a cleaning reagent tray is placed within the device with cleaning buffer and / or dilute bleach, and water. These cleaning cycles pump the cleaning solution into both the pump, sample fluid lines, and reagent fluid lines, remove and discard the cleaning solution, perform multiple cycles of washing with water, and then rinse the system with air.

[0173] New methods for the system integration of fluidics and microscopy enabling CISITS The new CISITS method requires an image acquisition system or microscope that can rapidly acquire images of the sample at sub-micron resolution over the depth of the sample for proper dot separation during combinatorial array determination. A plurality of different microscopes and microscope styles can be compatible with the CISITS fluid system, including confocal microscopy, spinning disk confocal microscopy, light sheet microscopy, lattice light sheet microscopy, and widefield microscopy, depending on whether combinatorial array determination with CISITS (greater requirements for resolution such that individual amplicons are resolved) or continuous array determination with CISITS (fewer requirements for resolution) is being performed.

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

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

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

[0177] In one example, the microscope is in an OEM format that allows microscope components to be more directly integrated with an automated fluid system, shipped in an integrated form, and removes unnecessary mechanical parts that are typically part of consumer products. The OEM microscope format allows for a more compact enclosure of the automated system. The direct integration facilitates the use of dedicated hardware for CISITS automation that interfaces directly with the imaging components of the microscope. These include, for example, repair of reagent leaks and detection at the objective or objective turret. In particular, new methods for large or multiple area acquisition for CISITS are mentioned, such as the automatic management of the liquid immersion interface between the objective lens and the sample chamber glass. Automatic management of the immersion interface, for example, of water, allows for large stage travel distances without losing immersion and proper refractive index matching, provides the ability for collection of whole sections, and importantly, allows the objective lens head to be converted to multiple different wells without losing the water (or other) immersion interface.

[0178] New software and new methods for regulating the automated alignment process Previous methods for automated STARmap alignment described firmware code that controls individual fluid components and suggested that this firmware code was adjusted by subsequent custom software on a controller computer.

[0179] The new CISITS method extends this process to a multi-level, hierarchical model of abstraction via system component control software with compatibility for parallel operation, integrates the array determination protocol into a single series of modular commands that accept a parameter file specifying the relevant details of array determination (from combinations to successive array determinations and variations of those protocols), provides a user interface for specifying, starting, pausing, and stopping array determination execution, and for starting wash execution, provides a system dashboard accessible via a network that displays criteria including array determination progress and quality assurance, and provides a method for a data management system to accommodate large datasets that would otherwise block array determination progress due to space constraints. These new methods provide means for the rapid, automated, and continuous acquisition of CISITS data.

[0180] Software Method for Hierarchical Abstraction and System Control The lowest level of firmware code is the individual controller for each device within the fluid system, such as 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 for operating these hardware components according to input parameters. This microcontroller code is interfaced via a continuous connection with a computer capable of image acquisition. The CISITS software itself, executed on the computer, is hierarchical and provides multiple abstractions regarding the control of various hardware components. In one example, this software is Py It is implemented in the Python programming language. The low-level module provides an object-oriented model for the programmatic interface connection with the hardware microcontroller / Arduino. The initialization of this object (the "robot") opens a continuous connection with the microcontroller, provides a handle for subsequent communication via the continuous interface connection, and resets the microcontroller device. Additionally, the initialization sets the basic parameters for the operation of the device that are invariant for different array determination protocols, such as the maximum pump movement speed, the maximum volume, and various time constants for fluid equilibration. The way to provide the lowest level of abstraction in this object is the send command method, which communicates a properly formatted continuous command string to the microcontroller and triggers the appropriate control macro. Commands to the microcontroller to perform individual operations of the fluid system, including getting and setting the temperature of the cooler, waiting until the cooler reaches the target temperature, moving the contents of the pump to waste, filling a specified volume of the input reagent into a specified sample, discharging a specified volume of a specified sample well to waste, and moving the reagent between reagent containers, are coordinated in a higher-level way of the object. The final key method of the object is to close the continuous connection to the microcontroller. By establishing and maintaining this continuous connection with the robot object, the software blocks access to other programmatic threads to the microcontroller and ensures that a single thread can adjust the fluid hardware at a time.

[0181] This control object is initialized and the method is called by an array determination module that provides a higher-level framework for the specifications of the array determination protocol. In one example of a CISITS software method, the array determination commands are executed sequentially for each individual operation, and the array determination procedure is organized in an "execution blocking" paradigm where the program waits until each command is completed. This provides a convenient and robust method for preventing competing commands from acting on the system simultaneously, especially during incubation times, as the program can be blocked from execution for only the allotted time. In this paradigm, the progression through the CISITS protocol, including reagent addition and removal and incubation times, occurs strictly sequentially after the execution of a program like a script. Multiple washings or additions of reagents occur within the program for all loops, and these are summarized in loops within the program over the course of the array determination rounds.

[0182] In another example of a CISITS software method, the array determination control for parallel samples is executed as a multi-threaded application, where each individual processing thread acquires a lock on the array determination hardware and asynchronously executes the required array determination steps at one thread per sample well. In this mode of operation, imaging and fluid movement can occur simultaneously. In another example, the threads operate asynchronously but communicate to optimize the timing of the array determination operations and meet the strict incubation time constraints in 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 the 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 states of the system components. This paradigm is particularly suited to reciprocal or parallel operation of the sequencer across multiple samples, where not all samples are absolutely synchronized in their sequencing stages. For example, a sample incubation command may be executed such that a timer is set and checked for a given sample, and when the timer is completed, the program may continue with that sample. However, in the meantime, the state of the pump or valve is available and may be commanded to operate on other samples while the first sample is incubating.

[0184] The sequencing module is responsible for proper fluid transfer and accidental spills, etc. The sequencing loop or event loop also includes a check for an interface file communicating with the sequencing thread that indicates the sequencing is 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 stopped interface file, the sequencing module reports to the log that a pause was detected, resets the device, and exits. In general, when the sequencing device starts to run, it writes a file indicating that it is running, and deletes the file when it completes its run or when it is present.

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

[0186] Software method for specifying parameters of the CISITS array determination device operation The array determination module of the CISITS control software is parameterized by settings specific to a given array determination protocol, such as combinatorial or sequential array determination. This method is designed such that specification files for different array determination methods fully parameterize the commands of the array determination device module. The default settings provide sufficient robustness for the automatic execution of the array determination device, but the user interface provides an advanced mode in which these settings can be changed and saved. This model, which separates the parameters from the main array determination method, enables the recording, reproducibility, and reuse of parameters and simplifies the software to a common array determination module.

[0187] Software method for graphical user interface control of array determination The graphical user interface (GUI) is the main interface between the user and the CISITS sequencer. It displays the status of the sequencer during execution and provides buttons to pause or stop the operation of the sequencer. While the sequencing module is running on a separate thread, it repeatedly checks the file in that directory indicating whether the sequencing should be paused or completely stopped, and enables communication between the user interface thread, which is otherwise independent, and the sequencing thread when sequencing is started. When no sequencing or washing is in progress, the GUI provides buttons to start a new sequencing run and to start a new washing run. This also indicates whether washing needs to be performed based on the elapsed time since the last CISITS sequencing run was carried out. When washing is started (the wash button is pressed), the GUI places the wash reagent tray and displays a message to the user to wash the sample container within the sequencer. When the user presses a button to confirm that these are in the correct positions, the wash cycle proceeds and the user is notified on the GUI of any errors. When sequencing is started (the sequence button is pressed), the user is provided with a text input box for the experiment name, a drop-down list to select either combinatorial sequencing or continuous sequencing, and a drop-down list indicating the number of rounds to be performed. The user can also click a button to display detailed options including a text input box showing the default sequencing parameters for combinatorial or continuous sequencing, can load parameters from a saved parameter file, and can enter different values and a file name to save a new set of custom sequencing parameters. The user can set a directory or location to transfer data, including an option to set up automatic transfer to cloud storage. After entering relevant information in the GUI, press the Continue button to start array determination or the Cancel button to return to the home screen. Pressing the Start button, the GUI briefly opens an interface object to the fluid system, checks whether the cooler is already cold enough, and if the cooler is not yet cold, starts cooling before disconnecting from the fluid system. Next, the GUI instructs the user to the next screen throughout the remaining array determination setup procedure, including setting up imaging parameters and filling the machine with reagents. Finally, the Start button can be pressed to start the array determination operation, and the array determination module is called as a thread with the input or selected parameters. At that point, the GUI ensures that the local server thread is being executed to provide a network-accessible array determination device status dashboard, and that the data transfer thread is being executed. After completing the main array determination operation and pre-washing, the GUI communicates to the user a message to replace the sample container with a wash sample container, and clicking the Continue button performs the final wash cycle of the array determination device.

[0188] New Software Method for System Status Dashboard The initialization of sequencing by CISITS software also launches a web server thread that functions 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 several samples. This includes data on which round of CISITS is being performed, elapsed time, estimated time until completion, data on completion times from round to round, data on image acquisition times, and quality control criteria. This depends, for example, on combinatorial sequencing, the distribution of the number of dots detected in the first round per field of view per region, the median dot size, the distribution of dots across color channels, and the registration distance between rounds, for example, sequencing, for example, the correlation of signals across channels within a round, or the correlation of signals between rounds within a color channel. To prevent unauthorized network access to this dashboard, the user needs to log in and view it on the dashboard, the server limits the number of network requests it can receive from IP address blocking to reduce denial-of-service attacks on the acquisition computer, and the dashboard does not provide any control over the sequencing device. Data from the dashboard is provided by the sequencing device module, which records operations and times via, for example, log files and files saved via an external processing pipeline, and when they become available, records the quality control data of the computer on the sequencing data and provides the network location where the quality control data can be obtained to the dashboard.

[0189] New Software Methods for the Acquisition and Management of CISITS Data Large-area CISITS array determination rounds, multiple array determination rounds, and parallel array determination of multiple samples or multiple chambers all produce data per sample per array determination round that can fill a large portion of a large hard drive. For example, a single tile microscopy acquisition of an area of interest of a CISITS sample can be in the range of hundreds of gigabytes to several terabytes. Therefore, software methods are essential to actively move the data from the acquisition hard drive onto a very large local storage drive pool, or onto a large buffer drive, and subsequently onto a large network storage drive or cloud storage for subsequent downstream processing. These software methods can be run as independent threads that check for available data and move them from the acquisition drive to the storage destination. These methods are essential for large-scale and high-throughput sequencing as failure to manage the data properly can rapidly lose space on the acquisition drive and potentially halt the sequencing.

[0190] New software methods for integration with microscope platforms The CISITS software method integrates fluid system operation with the operation of a microscope platform. In some examples, the microscope platform provides a stand-alone image acquisition GUI that can be used to set desired imaging parameters, fields of view, tiles, and positions, and is triggered or initiated by serial, TTL, REST, or other API commands from an array determination software module. In this case, the array determination module automatically communicates with the image acquisition software to initiate the user-specified image protocol at the correct time within the array determination chemistry protocol and waits for the acquisition of that sample to complete before initiating subsequent image acquisition or procedures. Alternatively, as in the case of parallel sample well operation, the software initiates image acquisition while periodically querying the image acquisition software about the completion status of ongoing acquisitions before starting acquisitions placed in a new queue and can proceed with other fluid operations on other samples. In other examples, particularly when the microscope platform is directly integrated by a fluid system, the CISITS software itself integrates sufficient microscope functionality within the GUI for the user to set desired image acquisition parameters before array determination is initiated. 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 acquisition software of the microscope system. Minimal CISITS software integration with the microscope platform includes setting of pre-modification of the acquisition file name, execution of saved imaging protocols in the microscope system, acquisition of the completion status of acquisitions, parallel operation of the array determination device across multiple sample wells, and acquisition and setting of stage positions. With CISITS software controlling array determination across multiple sample regions, the user can specify the starting positions of each different region to be imaged and associate those regions with parallel well array determination or multiple positions to be imaged sequentially within a single chamber.This is achieved during imaging settings where software queries the microscope system for stage positions and stores the stage positions as a list of acquisition start positions for a given sample, or as a list of acquisition start positions in a sample chamber with a different parallel order of an external list.

[0191] Use Insitu nucleic acid sequencing techniques from laboratory scale to clinical and industrial scale are described herein. Sample types for these techniques can include layers of cultured cells, cultured organoids, or sections of cultured organoids, as well as animal biopsy samples or postmortem animal tissues. This 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 within cells or tissues, changes in RNA expression across experimental conditions, and can identify the spatial expression of genes in cells expressing other orthogonal markers, including being used in gene screening or engineering screening.

[0192] Advantages and improvements over existing methods, devices, or materials Compared to the aforementioned STARmap method, CISITS combines chemical improvements to SNR and process robustness, as well as improvements in automation (hardware and software) to throughput and robustness, enabling improved robustness and high-throughput intact tissue sequencing.

[0193] Description of research or development funded by the federal government The present invention was made with government support under Contract W911NF-14-2-0013 awarded by the Defense Advanced Research Projects Agency and Contracts DA042012 and MH075957 awarded by the National Institutes of Health. The United States government has certain rights in this invention.

Claims

**Claim 1**: A method for in situ gene sequencing of target nucleic acids in cells in fixed and permeabilized intact tissues, comprising: (a) contacting a fixed and permeabilized intact tissue sample with at least a pair of oligonucleotides under conditions that allow specific hybridization, said pair of oligonucleotides comprising a first oligonucleotide and a second oligonucleotide, said second oligonucleotide comprising a barcode sequence; (b) adding a ligase to ligate the 5' and 3' ends of said second oligonucleotide to generate a closed nucleic acid ring; (c) incubating said fixed and permeabilized intact tissue sample with a buffer containing DNA polymerase; (d) performing rolling circle amplification by contacting said fixed and permeabilized intact tissue sample with deoxynucleotide triphosphates to generate one or more amplicons, wherein said ligated second oligonucleotide functions as a template and said first oligonucleotide functions as a primer for said DNA polymerase; (e) embedding said fixed and permeabilized intact tissue sample in a hydrogel either before or after any one of steps (a) to (d); (f) cross-linking said one or more amplicons to said hydrogel; (g) purifying said hydrogel; (h) contacting said one or more hydrogel-embedded amplicons having said barcode sequence with a read primer and a fluorescently labeled probe, wherein said fluorescently labeled probe comprises an oligonucleotide comprising a first thiol group covalently bonded to a fluorophore containing a second thiol group through a disulfide bond between the first thiol group and the second thiol group; (i) ligating said read primer and said fluorescently labeled probe; (j) contacting said hydrogel with an anti-fading buffer containing an antioxidant; (k) imaging said one or more hydrogel-embedded amplicons; (l) contacting said hydrogel with a reducing agent; (m) removing said fluorophore from said hydrogel. (n) repeating steps (h) to (m); A method comprising the steps of: **Claim 2** The method according to claim 1, wherein the antioxidant is N-propyl gallate. **Claim 3** The method according to claim 1 or 2, wherein endogenous RNA is first bound to the hydrogel prior to step (a) for the fixed and permeabilized intact tissue sample. **Claim 4** The method according to any one of claims 1 to 3, wherein the purification of the hydrogel is carried out before or after any one of steps (a) to (d). **Claim 5** The method according to any one of claims 1 to 4, wherein the contact with the fixed and permeabilized intact tissue comprises hybridizing a plurality of oligonucleotide primers having specificities for different target nucleic acids. **Claim 6** The method according to any one of claims 1 to 5, wherein the deoxynucleotide triphosphates include amine-modified deoxynucleotide triphosphates, and optionally, the amine-modified deoxynucleotide triphosphates include N-hydroxysuccinimide acrylate moiety modification. **Claim 7** The method according to any one of claims 1 to 6, wherein the intact tissue has a thickness of 5 to 20 μm or 50 to 200 μm. **Claim 8** The method according to any one of claims 1 to 7, further comprising slicing the fixed and permeabilized intact tissue and flatly arranging the slices of the fixed and permeabilized intact tissue on a surface, and optionally, a metal ring is pressed down on the slices and used to move the slices flat on the surface. **Claim 9** The method according to any one of claims 1 to 8, wherein the reducing agent is TCEP. **Claim 10** The method according to any one of claims 1 to 9, wherein the hydrogel is contacted with the reducing agent for 30 minutes in step (l). **Claim 11** The method according to any one of claims 1 to 10, further comprising exposing the lead primer and the fluorescently labeled probe to a stripping buffer containing 80% formamide before step (l). **Claim 12** Each of the first oligonucleotide and the second oligonucleotide includes a first complementary region, a second complementary region, and a third complementary region, the second oligonucleotide further includes a barcode sequence, 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, the second complementary region of the second oligonucleotide is complementary to a second portion of the target nucleic acid, and the first complementary region of the first oligonucleotide is adjacent to the second complementary region of the second oligonucleotide. The method according to any one of claims 1 to 11.

13. The incubation in step (c) is carried out for a sufficient time to allow uniform diffusion of DNA polymerase throughout the immobilized and permeabilized intact tissue sample before performing rolling circle amplification. The method according to any one of claims 1 to 12.

14. The ligation in step (i) occurs only when both the read primer and the fluorescently labeled probe are complementary to adjacent sequences of the same amplicon. The method according to any one of claims 1 to 13.

15. A fluid system comprising (a) a device comprising an unpressurized sample chamber, optionally a well of a multi-well plate or a slide chamber, and (b) a lid that covers the top of the sample chamber. (c) An interface tube, wherein a first end of the interface tube is held by the lid, and a second end of the interface tube is positioned sufficiently close to a bottom edge of the sample chamber such that the liquid in the sample chamber remains in contact with a bottom of the interface tube as long as liquid remains in the sample chamber, and is positioned over an immobilized and permeabilized intact tissue sample in the sample chamber, and optionally, the interface tube is disposed at a polar angle with respect to a plane of the immobilized and permeabilized intact tissue sample, the interface tube; (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 onto the immobilized and permeabilized intact tissue sample in the sample chamber from the second end of the interface tube, the fluid line; (e) A pump, optionally, the pump being a syringe pump, the pump; (f) An imaging system; (g) A processor unit configured to perform the method according to any one of claims 1 to 14 A fluid system comprising.

16. The fluid system according to claim 15, further comprising a cryostat for maintaining the sample chamber at cryogenic temperatures, a multi-directional selector valve interfaced with the pump, or 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-directional selector valve or combinations thereof.

17. The fluid system according to claim 16, further comprising an adjustable stage for 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.

18. The fluid system according to any one of claims 15 to 17, further comprising a container surrounding the fluid system, the container blocking ambient light, and optionally, the container surrounding the fluid system being an acoustic attenuation container.

19. The fluid system according to any one of claims 15 to 18, further comprising means for cryosectioning the tissue sample.

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