In-situ gene sequencing method

Abstract

Devices, methods, and systems are provided for in situ gene sequencing of a target nucleic acid in a cell within intact tissue. The invention also provides methods of screening candidate agents to determine whether the candidate agents are capable of modulating gene expression of nucleic acids in cells within intact tissue.

Description

This application is a divisional application of the invention application with an application date of April 4, 2019, Chinese application number 201980038374.3, and invention name “In situ gene sequencing method”. Cross-references

[0001] This patent application claims the benefit of U.S. Provisional Application No. 62 / 655,052, filed on April 9, 2018 Patent application, U.S. Provisional Patent Application No. 62 / 687,490 filed on June 20, 2018, and U.S. Provisional Patent Application No. 62 / 808,159 filed on February 20, 2019; the contents of the above patents are incorporated herein by reference in their entirety. introduce

[0002] In biological tissues, functional diversity stems from morphological diversity and, in part, from the complexity of cell-specific gene expression, which determines the unique three-dimensional molecular anatomy and cellular properties of each tissue. In situ transcriptome analysis tools are now available for mapping spatial maps of gene expression with subcellular resolution, which may be suitable for exploring these tissue structure-function relationships, including multiplex in situ RNA hybridization and in situ RNA sequencing. Existing in situ sequencing methods face challenges in achieving enzymatic reactions in dense and complex tissue environments, and current sequencing efficiency is low, but this in-tissue sequencing method has great potential value; compared with hybridization-based multiplex analysis / readouts (which use multiple polynucleotide probes to encode gene identity and sequencing), this in-tissue sequencing method can operate at single nucleotide resolution, so the amount of information it provides is essentially greatly increased. However, due to fundamental limitations in sensitivity, fidelity, and scalability required to achieve throughput in tissues (e.g., mammalian brain), existing sequencing methods are not applicable to 3D intact tissues. For example, the mammalian brain is composed of a variety of complex cell types, and its diversity is critical for functions that depend on differential gene expression and circuit-specific anatomical structures. It is difficult to obtain high-content gene expression information while retaining 3D localized anatomical structures at cellular resolution, which limits the comprehensive understanding of brain structure and function. The present invention solves the above problems and provides corresponding advantages. Summary of the invention

[0003] The present invention provides an apparatus, method and system for in situ gene sequencing of a target nucleic acid in cells in intact tissues. The present invention also provides a method for screening a candidate agent to determine whether the candidate agent can regulate the gene expression of a nucleic acid in cells in intact tissues.

[0004] The present invention provides a method for in situ gene sequencing of a target nucleic acid in a cell in an intact tissue, the method comprising: (a) contacting a fixed permeabilized intact tissue with at least one pair of oligonucleotide primers under conditions that allow specific hybridization to occur, wherein the primer pair comprises a first oligonucleotide and a second oligonucleotide; wherein the first oligonucleotide and the second oligonucleotide each comprise a first complementary region, a second complementary region, and a third complementary region; wherein the second oligonucleotide further comprises a barcode sequence; wherein the first complementary region of the first oligonucleotide is complementary to a first portion of the target nucleic acid, wherein the second complementary region of the first oligonucleotide is complementary to the first complementary region of the second oligonucleotide, wherein the third complementary region of the first oligonucleotide is complementary to the third complementary region of the second oligonucleotide, wherein the second complementary region of the second oligonucleotide is complementary to a second portion of the target nucleic acid, and wherein the first complementary region of the first oligonucleotide is complementary to the third complementary region of the second oligonucleotide; The second complementary regions of the two oligonucleotides are adjacent; (b) a ligase is added to connect the second oligonucleotide and generate a closed nucleic acid circle; (c) rolling circle amplification is performed in the presence of a nucleic acid molecule, wherein the operation comprises using the second oligonucleotide as a template and using the first oligonucleotide as a primer to cause 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 connection, wherein the primer pair comprises a third oligonucleotide and a fourth oligonucleotide, wherein the connection is achieved only when the third oligonucleotide and the fourth oligonucleotide are connected to the same amplicon; (f) repeating step (e); and (g) imaging the one or more hydrogel-embedded amplicons to determine in situ gene sequencing of target nucleic acids in cells within intact tissues. In some cases, the primer pair is heat denatured before contacting the sample. In some cases, the cells are present in a cell population. In some cases, the population of cells includes multiple cell types.

[0005] The present invention also provides a method for screening a candidate agent to determine whether the candidate agent can regulate gene expression of a nucleic acid in a cell in an intact tissue, the method comprising: (a) contacting a fixed permeabilized intact tissue with at least one pair of oligonucleotide primers under conditions that allow specific hybridization to occur, wherein the primer pair comprises a first oligonucleotide and a second oligonucleotide; wherein the first oligonucleotide and the second oligonucleotide each comprise a first complementary region, a second complementary region, and a third complementary region; wherein the second oligonucleotide further comprises a barcode sequence; wherein the first complementary region of the first oligonucleotide is complementary to a first portion of the target nucleic acid, wherein the second complementary region of the first oligonucleotide is complementary to the first complementary region of the second oligonucleotide, wherein the third complementary region of the first oligonucleotide is complementary to the third complementary region of the second oligonucleotide, wherein the second complementary region of the second oligonucleotide is complementary to a second portion of the target nucleic acid, and wherein the first complementary region of the first oligonucleotide is adjacent to the second complementary region of the second oligonucleotide; (b) adding a ligase to connect the second oligonucleotide and generate a closed nucleic acid circle; (c) ligating the nucleic acid to the target nucleic acid; and (d) ligating the nucleic acid to the target nucleic acid. (e) contacting the one or more hydrogel-embedded amplicons having a barcode sequence with a pair of primers under conditions that allow ligation, wherein the primer pair comprises a third oligonucleotide and a fourth oligonucleotide, wherein ligation is achieved only when the third oligonucleotide and the fourth oligonucleotide are ligated to the same amplicon; (f) repeating step (e); (g) imaging the one or more hydrogel-embedded amplicons to determine in situ gene sequencing of target nucleic acids in cells within intact tissues; 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 the at least one candidate agent relative to the expression level of the target nucleic acid in the absence of the at least one candidate agent indicates that the at least one candidate agent regulates the gene expression of the nucleic acid in cells within intact tissues. In some cases, the primer pair is heat denatured prior to contacting the sample. In some cases, the cell is present in a cell population. In some cases, the cell population includes multiple cell types.

[0006] The present invention also provides the equipment used according to the method described herein. In some embodiments, a fluid system for automatically implementing the method described herein is provided, thereby allowing continuous operation. In some embodiments, the system includes a fluid device, a processor configured to implement the method described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figures 1A-1E Depicted is a schematic diagram of the Spatially Resolved Transcript Amplicon Read Mapping (STARmap) method described herein, which involves in situ RNA sequencing for spatial transcriptomics within a 3D tissue environment.

[0008] Figure 2A-2I Specific amplification of nucleic acids based on intramolecular ligation (SNAIL) probes for high-quality RNA imaging and instantiation in mouse brain are described.

[0009] Figures 3A-3H The hydrogel histochemistry method (HTC) used in STARmap to reduce background noise and immobilize amplicons is described.

[0010] Figures 4A-4L Aspects of sequencing by dynamic annealing and ligation (SEDAL), the low-background, error-corrected in situ sequencing component of STARmap, are described.

[0011] Figures 5A-5N The classification of cell types in the primary visual cortex using the methods described herein is described.

[0012] Figures 6A-6C The data processing pipeline used for STARmap is described.

[0013] Figures 7A-7D Gene expression information suitable for STAR mapping of inhibitory and excitatory subclusters is described.

[0014] Figures 8A-8C Sub-clusters of non-neuronal cell types are depicted.

[0015] Figures 9A-9G The lack of batch effects in achieving reproducibility of cell clustering using the methods described here is described.

[0016] Figures 10A-10D Behavioral experience using the methods described herein to detect and quantify cell type-specific regulation of activity regulated genes (ARGs) is described.

[0017] Figures 11A-11C The dark-treated samples, light-treated samples and ARG expression information obtained using the method described in this article are described.

[0018] Fig.12 Experimental flow chart describing STARmap for thin and thick tissue sections.

[0019] Figures 13A-13I Describes the three-dimensional organization of cell types in the visual cortex.

[0020] Figures 14A-14B Continuous SEDAL readouts and expression of multiple marker genes are depicted.

[0021] Figures 15A-15C 2D nearest neighbor analysis and cross-method validation of short-range inhibitory clustering in mouse primary visual cortex are described.

[0022] Figures 16A-16E Describes the scalability of STARmap.

[0023] Fig.17 Describes the correlation of neuronal types identified in STARmap with published single-cell RNA sequencing results.

[0024] Figures 18A-18F Gene expression analysis of cell type subclusters in the medial prefrontal cortex (mPFC) using the methods described herein is described.

[0025] Figures 19A-19C Described is the reproducible determination of cellular clustering and spatial organization of the mPFC using the methods described herein.

[0026] Figures 20A-20C Described is the analysis of 1020 genes in mouse hippocampal cell cultures in 6 rounds of sequencing using the methods described herein.

[0027] Figures 21A-21C Described is a gene expression analysis of 1020 genes in mouse primary visual cortex using the methods described herein.

[0028] Figures 22A-22D Described is the reproducibility determination and cross-method comparison of measurements of 1020 genes in the mouse primary visual layer using the methods described herein.

[0029] Figures 23A-23D Experimental flow chart and cost estimates for STARmap applicable to thin and thick tissue sections are described.

[0030] Figures 24A-24B Depicted are floor plans of exemplary fluid systems for implementing the methods described herein. Implementation

[0031] The present invention provides an apparatus, method and system for in situ gene sequencing of a target nucleic acid in cells in intact tissues. The present invention also provides a method for screening a candidate agent to determine whether the candidate agent can regulate the gene expression of a nucleic acid in cells in intact tissues.

[0032] In some embodiments, the disclosed method for 3D whole tissue RNA sequencing in the brain and other organs, referred to as spatially resolved transcript amplicon read map mapping (STARmap), includes integrating an improved ligation sequencing workflow, specific signal amplification, and hydrogel tissue chemistry ( Figure 1A ). In some aspects, STARmap can map cellular-resolution expression by sequencing 160 different genes in all cells within a mouse visual cortex slice. In some aspects, STARmap can identify different anatomically and molecularly resolved cell types within cortical layers (including interneuron and glial subtypes) and quantify the expression of activity-regulated genes, which varies with visual stimulation, spatial location, and molecularly defined cell type. In some aspects, STARmap can quantify more than 30,000 cells in a cubic millimeter volume, showing a gradient distribution of excitatory neuronal subtypes in stark contrast to the 3D clustering pattern of inhibitory neurons.

[0033] In some embodiments, in situ synthetic hydrogels are integrated with intracellular delivery interfaces (coupled with natural biomolecules), which can transform tissues (starting from the interior of their constituent cells) into a new state suitable for high-resolution volumetric imaging and analysis (compatible with a variety of molecular phenotyping applicable to proteins, nucleic acids and other targets). For example, synthetic hydrogels have been used to accommodate enzymatic reactions (including DNA sequencing) and are well known in the art, including but not limited to the technology disclosed in WO2014 / 025392, the contents of which are incorporated herein by reference. In some embodiments, biological tissues can be converted into hydrogel-embedded forms compatible with the generation, retention and functional expression of RNA-derived complementary DNA (cDNA). In these aspects, 3D in situ sequencing can be performed in the tissue hydrogel formulation, thereby taking advantage of the key accompanying properties of optical transparency, reducing background noise, increasing diffusion rate, and improving mechanical stability.

[0034] The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and refer to amino acids of any length in polymeric form, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. The term "polypeptide" includes fusion proteins, including but not limited to fusion proteins with heterologous amino acid sequences; fusion proteins with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunolabeled proteins, etc. The term "polypeptide" includes polypeptides that meet the following requirements, namely, include one or more of a fatty acid portion, a lipid portion, a sugar portion, and a carbohydrate portion. The term "polypeptide" includes post-translationally modified polypeptides.

[0035] The term "target nucleic acid" as used herein refers to any polynucleotide nucleic acid molecule (e.g., DNA molecules, RNA molecules, modified nucleic acids, 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, mRNA precursor, etc.) in the cell environment. Therefore, a suitable target nucleic acid can be an unspliced ​​RNA (e.g., mRNA precursor, mRNA), a partially spliced ​​RNA, or a completely spliced ​​RNA, etc. The target nucleic acid of interest can be expressed to varying degrees in a cell population, i.e., with different abundances, wherein the method of the present invention allows analysis and comparison of expression levels of nucleic acids (including but not limited to RNA transcripts) in individual cells. The target nucleic acid can also be a DNA molecule, for example, denatured genomic DNA, viral DNA, plasmid DNA, etc. For example, the methods can be used to detect copy number variation, e.g., in a cancer cell population, where the target nucleic acid is present at varying abundances in the genomes of cells within the population and in virally infected cells, in order to determine viral load and kinetic characteristics, among other things.

[0036] The terms "oligonucleotide," "polynucleotide," and "nucleic acid molecule" are used interchangeably herein to refer to nucleotides of any length in polymeric form, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, 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 derived nucleotide bases. The backbone of the polynucleotide may include sugar and phosphate groups (commonly found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the polynucleotide may include a polymer composed of synthetic subunits (e.g., phosphoramidites and / or phosphorothioates), and thus may be an oligodeoxynucleoside phosphoramidate or a mixed phosphoramidate-phosphodiester oligomer. Peyrottes et al. (1996), Nucleic Acids Research, 24: 1841-1848; Chaturvedi et al. (1996), Nucleic Acids Research, 24: 2318-2323. The polynucleotide may include one or more L-type nucleosides. The polynucleotide may include modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracil, other sugars, linking groups (such as fluororibose and thioesters) and nucleotide branches. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may be modified to include N3'-P5' (NP) phosphoramidate, morpholino phosphorodiamidate (MF), locked nucleic acid (LNA), 2'-O-methoxyethyl (MOE) or 2'-fluoroarabinose nucleic acid (FANA), which may enhance the resistance of the polynucleotide to nuclease degradation (see, for example, Faria et al. (2001), Nature Biotechnology, 19: 40-44; Toulme (2001), Nature Biotechnology, 19: 17-18). The polynucleotide may be further modified after polymerization, such as by conjugating with a labeling component. Other types of modifications included in this definition are capping, replacement of one or more naturally occurring nucleotides with analogs, and the introduction of a method for attaching a polynucleotide to a protein, a metal ion, a labeling component, other polynucleotides or a solid support. Immunomodulatory nucleic acid molecules can be provided in various formulations, for example, in association with liposomes, microencapsulation, etc., as described herein. The polynucleotides used in amplification are typically single-stranded polynucleotides to maximize amplification efficiency, but can also be double-stranded polynucleotides. - If it is a double-stranded polynucleotide, the polynucleotide can be treated to separate its double strands before being used to prepare an extension product. - This denaturation step is usually affected by high temperatures, but can also be performed using alkali and then neutralized.

[0037] "Subject" or "individual" or "patient" refers to any subject in need of treatment. Human subjects are of particular interest. Other subjects may include non-human primates, cattle, sheep, goats, dogs, cats, birds (e.g., chickens or other poultry), guinea pigs, rabbits, rats, mice, horses, etc. Of note are subjects who have or are susceptible to brain damage.

[0038] Before further describing the present invention, it should be understood that the present invention is not limited to the specific embodiments described, as there will certainly be differences in actual implementation. It should also be understood that the terms used in this patent are only used to describe specific embodiments and are not intended to limit the inventive concept. The scope of the present invention will only be limited by the appended claims.

[0039] Where a numerical range is provided, it is to be understood that each intermediate value between the upper and lower limits of the range and any other specified value or intermediate value within the range are included within the scope of the present invention. Unless the context clearly specifies otherwise, each intermediate value should be as low as one-tenth of the lower limit unit. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges, and are also included within the present invention, subject to the requirements of any specifically excluded limits within the range. Where the range includes one or two limits, the range excluding any one or two of those included limits is also included within the present invention.

[0040] Unless otherwise defined, all technical and scientific terms used in this patent have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, preferred methods and materials are described below. All publications mentioned in this patent are incorporated herein by reference to disclose and describe the methods and / or materials related to the cited publications.

[0041] It must be noted that, as used in this patent 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 "the oligonucleotide" refers to one or more oligonucleotides and equivalents known to those skilled in the art, and so forth. It should also be noted that claims may be drafted to exclude any optional element. Therefore, this statement is intended to serve as an antecedent basis for the use of specialized terminology such as "solely," "only," etc., in connection with reciting claim elements, or for the use of a "negative" limitation.

[0042] It should be understood that certain features of the invention described in the context of separate embodiments for clarity may also be presented in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for brevity may also be presented individually or in any suitable sub-combination. All combinations of embodiments related to the invention are expressly contemplated by the invention and disclosed herein as if each combination were individually and expressly disclosed. In addition, all sub-combinations of the various embodiments and their constituent parts are also expressly contemplated by the invention and disclosed herein as if each sub-combination were individually and expressly disclosed.

[0043] The publications discussed in this patent are provided solely for their disclosure prior to the filing date of the patent. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. method

[0044] The methods disclosed herein include image-based in situ nucleic acid (DNA and / or RNA) sequencing technology that can be used to perform spatially resolved, highly multiplexed gene detection at the subcellular and cellular levels through improved ligation sequencing processes, specific signal amplification, hydrogel tissue chemistry methods that transform biological tissues into transparent sequencing chips, and related data analysis pipelines (collectively referred to as "Spatially Resolved Transcript Amplicon Read Mapping (STARmap)"). In some embodiments, STARmap defines a platform for 3D in situ transcriptome analysis, which is achieved through improved cDNA library preparation, sequencing, and hydrogel tissue chemistry methods.

[0045] As described above, the methods disclosed herein include a method for in situ genetic sequencing of a target nucleic acid in a cell in an intact tissue, the method comprising: (a) contacting a fixed permeabilized intact tissue with at least one pair of oligonucleotide primers under conditions that allow specific hybridization to occur, wherein the primer pair comprises a first oligonucleotide and a second oligonucleotide; wherein the first oligonucleotide and the second oligonucleotide each comprise a first complementary region, a second complementary region, and a third complementary region; wherein the second oligonucleotide further comprises a barcode sequence; wherein the first complementary region of the first oligonucleotide is complementary to a first portion of the target nucleic acid, wherein the second complementary region of the first oligonucleotide is complementary to the first complementary region of the second oligonucleotide, wherein the third complementary region of the first oligonucleotide is complementary to the third complementary region of the second oligonucleotide, wherein the second complementary region of the second oligonucleotide is complementary to a second portion of the target nucleic acid, and wherein the first complementary region of the first oligonucleotide is complementary to the third complementary region of the second oligonucleotide The invention relates to a method for preparing a nucleic acid molecule comprising: (a) preparing a nucleic acid molecule comprising: (i) preparing a nucleic acid molecule comprising: (i) preparing a nucleic acid molecule comprising: (a) preparing a nucleic acid molecule comprising: (b) preparing a nucleic acid molecule comprising: (c) preparing a nucleic acid molecule comprising: (d) preparing a nucleic acid molecule comprising: (i) preparing a nucleic acid molecule comprising: ...

[0046] The method disclosed herein also provides a method for screening a candidate agent to determine whether the candidate agent can regulate gene expression of a nucleic acid in a cell in an intact tissue, the method comprising: (a) contacting a fixed permeabilized intact tissue with at least one pair of oligonucleotide primers under conditions that allow specific hybridization to occur, wherein the primer pair comprises a first oligonucleotide and a second oligonucleotide; wherein the first oligonucleotide and the second oligonucleotide each comprise a first complementary region, a second complementary region, and a third complementary region; wherein the second oligonucleotide further comprises a barcode sequence; wherein the first complementary region of the first oligonucleotide is complementary to a first portion of the target nucleic acid, wherein the second complementary region of the first oligonucleotide is complementary to the first complementary region of the second oligonucleotide, wherein the third complementary region of the first oligonucleotide is complementary to the third complementary region of the second oligonucleotide, wherein the second complementary region of the second oligonucleotide is complementary to a second portion of the target nucleic acid, and wherein the first complementary region of the first oligonucleotide is adjacent to the second complementary region of the second oligonucleotide; (b) adding a ligase to connect the second oligonucleotide and form a closed nucleic acid circle; (c) (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, wherein the primer pair includes a third oligonucleotide and a fourth oligonucleotide, wherein ligation is achieved only when the third oligonucleotide and the fourth oligonucleotide are ligated to the same amplicon; (f) repeating step (e); (g) imaging the one or more hydrogel-embedded amplicons to determine in situ gene sequencing of target nucleic acids in cells within intact tissues; and (h) detecting gene expression levels of the target nucleic acids, wherein a change in the expression level of the target nucleic acids in the presence of the at least one candidate agent relative to the expression level of the target nucleic acids in the absence of the at least one candidate agent indicates that the at least one candidate agent regulates gene expression of nucleic acids in cells within intact tissues.

[0047] In some aspects, compared with existing gene expression analysis tools, the method disclosed herein can shorten the processing time, increase multiplicity (up to 1000 genes), improve efficiency, improve sensitivity, reduce error rate, and increase the cell type of spatial resolution. In these aspects, the improved hydrogel tissue chemistry method can convert biological tissue into a nucleic acid with a hydrogel imprint compatible with in situ sequencing, which is an improved connection method sequencing process (SEDAL) suitable for in situ sequencing with reduced error rate. In some other aspects, the method disclosed herein includes spatial sequencing (e.g., reagents, chips or services) for biomedical research and clinical diagnosis (e.g., cancer, bacterial infection, viral infection, etc.) with single cell and / or single molecule sensitivity. Specific amplification of nucleic acid by intramolecular ligation (SNAIL)

[0048] In some embodiments, a component of STARmap includes an efficient method for generating cDNA libraries in situ using cellular RNA, which can be referred to as SNAIL, i.e., specific amplification of nucleic acids by intramolecular ligation. In certain embodiments, the method of the invention includes contacting fixed permeabilized intact tissue with at least one pair of oligonucleotide primers under conditions that allow specific hybridization to occur, wherein the primer pair includes a first oligonucleotide and a second oligonucleotide.

[0049] More generally, the nucleic acid present in the target cell in the tissue can be used as a scaffold for a complex assembly, the complex comprising a pair of primers, which are referred to herein as a first oligonucleotide and a second oligonucleotide. In some embodiments, contacting the fixed permeabilized intact tissue comprises hybridizing the primer pair to the same target nucleic acid. In some embodiments, the target nucleic acid is RNA. In these embodiments, the target nucleic acid can be mRNA. In other embodiments, the target nucleic acid is DNA.

[0050] The terms "hybridization" and "hybridization" as used herein refer to the formation of a complex between nucleotide sequences that are appropriately complementary to form a complex via Watson-Crick base pairing. In the case of a primer "hybridizing" with a target (template), such a complex (or hybrid) is sufficiently stable to function as a priming agent, such as that required to cause DNA polymerase to initiate DNA synthesis. It should be understood that the hybridization sequences do not need to have perfect complementarity in providing a stable hybrid. In many cases, a stable hybrid is formed in which the proportion of mismatched bases is less than about 10%, and loops consisting of four or more nucleotides are ignored. Therefore, the term "complementary" as used herein refers to an oligonucleotide that forms a stable duplex with its "complement" under assay conditions (usually with a homology of about 90% or more). SNAIL oligonucleotide primers

[0051] In the method of the present invention, the SNAIL oligonucleotide primer comprises at least a first oligonucleotide and a second oligonucleotide; wherein the first oligonucleotide and the second oligonucleotide each comprise a first complementary region, a second complementary region and a third complementary region; wherein the second oligonucleotide further comprises a barcode sequence; wherein the first complementary region of the first oligonucleotide is complementary to a first portion of the target nucleic acid, wherein the second complementary region of the first oligonucleotide is complementary to the first complementary region of the second oligonucleotide, wherein the third complementary region of the first oligonucleotide is complementary to the third complementary region of the second oligonucleotide, wherein the second complementary region of the second oligonucleotide is complementary to a second portion of the target nucleic acid, and wherein the first complementary region of the first oligonucleotide is adjacent to the second complementary region of the second oligonucleotide. In an alternative embodiment, the second oligonucleotide is a closed circular molecule, and the ligation step is omitted.

[0052] The present invention provides methods, wherein contacting the fixed permeabilized intact tissue comprises hybridizing a plurality of oligonucleotide primers specific for different target nucleic acids. In some embodiments, the method comprises a plurality of first oligonucleotides hybridizing with a target nucleotide sequence, including but not limited to 5 or more first oligonucleotides, for example, 8 or more, 10 or more, 12 or more, 15 or more, 18 or more, 20 or more, 25 or more, 30 or more, 35 or more. In some embodiments, the method of the present invention comprises a plurality of first oligonucleotides, including but not limited to 15 or more first oligonucleotides, for example, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more and up to 80 different first oligonucleotides, which hybridize with 15 or more, for example, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more and up to 80 different target nucleotide sequences. In certain embodiments, the method comprises a plurality of second oligonucleotides, including but not limited to 5 or more second oligonucleotides, for example, 8 or more, 10 or more, 12 or more, 15 or more, 18 or more, 20 or more, 25 or more, 30 or more, 35 or more. In certain embodiments, the method of the present invention comprises a plurality of second oligonucleotides, including but not limited to 15 or more second oligonucleotides, for example, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more and 80 different first oligonucleotides at the most, which are hybridized with 15 or more, for example, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more and 80 different target nucleotide sequences at the most. A plurality of oligonucleotide pairs can be used in the reaction, wherein one or more oligonucleotide pairs are specifically bound to each target nucleic acid. For example, two primer pairs can be used for a target nucleic acid to improve sensitivity and reduce variability. It is also contemplated that a plurality of different target nucleic acids are detected 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 are detected. The primers are typically denatured prior to use, which is typically accomplished 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.

[0053] In some embodiments, the primers are heat denatured prior to contacting the sample. In certain aspects, the melting temperature (T m) to minimize oligonucleotide ligation in solution. The "melting temperature" or "Tm" of a nucleic acid is 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 (e.g., by acid or base treatment, etc.). The T of a nucleic acid molecule m Depends on its length and its base composition. T of nucleic acid molecules rich in GC base pairs m Above the melting temperature of nucleic acid molecules having a large number of AT base pairs. m When the hybridization rate is below 1000, the separated complementary strands of nucleic acid will spontaneously reassociate or anneal to form a double-stranded nucleic acid. The highest nucleic acid hybridization rate occurs at T m Below about 25 degrees Celsius. T can be estimated using the following relationship m :T m =69.3+0.41(GC)% (Marmur et al. (1962), Journal of Mol. Biol., 5:109-118).

[0054] In certain embodiments, the plurality of second oligonucleotides include padlock probes. In certain embodiments, the probe includes a detectable label that can be measured and quantified. The terms "label" and "detectable label" refer to molecules that can be detected, including but not limited to radioisotopes, fluorescent agents, chemiluminescent agents, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin or haptens), etc. The term "fluorescent agent" refers to a substance or portion thereof that can display fluorescence within a detectable range. Specific examples of labels that can be used in conjunction with the present invention include, but are not limited to, phycoerythrin, Alexa dyes, fluorescein, YPet, CyPet, Cascade blue, allophycocyanin, Cy3, Cy5, Cy7, rhodamine, dansyl, umbelliferone, Texas Red, luminol, acridinium esters, biotin, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), firefly luciferase, Renilla luciferase, NADPH, β-galactosidase, horseradish peroxidase, glucose oxidase, alkaline phosphatase, chloramphenicol acetyltransferase, and urease.

[0055] In certain embodiments, the one or more first oligonucleotides and the second oligonucleotides are combined with different regions or target sites of the target nucleic acid. In a target site pair, each target site is different, and the target site is an adjacent site on the target nucleic acid (for example, the spacing with another site is usually no more than 15 nucleotide distances, for example, no more than 10,8,6,4 or 2 nucleotide distances), and can be a continuous site. The target site is usually present on the same chain of the target nucleic acid with the same orientation. In addition, the target site can also be selected relative to other nucleic acids present in the cell, to provide a unique binding site. The length of each target site is generally about 19 to about 25 nucleotides, for example, about 19 to 23 nucleotides, about 19 to 21 nucleotides or about 19 to 20 nucleotides. Select the first and second oligonucleotide pairs so that each oligonucleotide in the oligonucleotide pair has a similar melting temperature, so as to be combined with its homologous target site, for example, the T m It may be not less than about 50° C., not less than about 52° C., not less than about 55° C., not less than about 58° C., not less than about 62° C., not less than about 65° C., not less than about 70° C., or not less than about 72° C. The GC content of the target site is usually selected to be no more than about 20%, no more than about 30%, no more than about 40%, no more than about 50%, no more than about 60%, or no more than about 70%.

[0056] In certain embodiments, the first oligonucleotide includes the first, second and third complementarity regions. The target site of the first oligonucleotide may refer to the first complementarity region. As described above, the length of the first complementarity region of the first oligonucleotide may be 19-25 nucleotides. In certain aspects, the length of the second complementarity region of the first oligonucleotide is 3-10 nucleotides, including, for example, 4-8 nucleotides or 4-7 nucleotides. In certain aspects, the length of the second complementarity region of the first oligonucleotide is 6 nucleotides. In certain embodiments, the length of the third complementarity region of the first oligonucleotide is 6 nucleotides equally. In these embodiments, the length of the third complementarity region of the first oligonucleotide is 3-10 nucleotides, including, for example, 4-8 nucleotides or 4-7 nucleotides.

[0057] In some embodiments, the second first oligonucleotide includes the first, second and third complementary regions. The target site of the second oligonucleotide may refer to the second complementary region. As described above, the length of the second complementary region of the second oligonucleotide may be 19-25 nucleotides. In certain aspects, the length of the first complementary region of the first oligonucleotide is 3-10 nucleotides, including, for example, 4-8 nucleotides or 4-7 nucleotides. In some aspects, the length of the first complementary region of the first oligonucleotide is 6 nucleotides. In some aspects, the first complementary region of the second oligonucleotide includes the 5' end of the second oligonucleotide. In some embodiments, the length of the third complementary region of the second oligonucleotide is also 6 nucleotides. In these embodiments, the length of the third complementary region of the second oligonucleotide is 3-10 nucleotides, including, for example, 4-8 nucleotides or 4-7 nucleotides. In a further embodiment, 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.

[0058] In some aspects, the second oligonucleotide includes a barcode sequence, wherein the barcode sequence of the second oligonucleotide provides barcode information for identifying the target nucleic acid. The term "barcode" refers to a nucleic acid sequence used to identify a single cell or a subpopulation of cells. The barcode sequence can be connected 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 originated. The barcode sequence can be added to the target nucleic acid of interest during amplification with an oligonucleotide containing a region including a barcode sequence and a region complementary to the target nucleic acid, so that the barcode sequence is incorporated into the target nucleic acid product (i.e., amplicon) of the final amplification. organize

[0059] As described herein, the disclosed methods include in situ sequencing techniques of intact tissues, which are achieved by contacting at least fixed permeabilized intact tissues with at least one pair of oligonucleotide primers under conditions that allow specific hybridization to occur. Tissue samples suitable for use in conjunction with the methods described herein generally include any type of tissue sample collected from a living or dead subject, such as biopsy samples and autopsy samples, including but not limited to epithelial tissue, muscle tissue, connective tissue, and neural tissue. Tissue samples can be collected and processed using the methods described herein, and the samples can be analyzed microscopically immediately after processing, or they can be stored and analyzed microscopically at a later date (e.g., after being stored for a longer period of time). In some embodiments, the methods described herein can be used to preserve tissue samples so that they remain stable, accessible, and intact for later analysis. In some embodiments, the methods described herein can be used to analyze previously preserved or stored tissue samples. In some embodiments, the intact tissue includes brain tissue, such as a visual cortex slice. In some embodiments, the intact tissue is a thin slice having a thickness of 5-20 μm, including but not limited to, for example, 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, for example, 50-150 μm, 50-100 μm, or 50-80 μm.

[0060] Aspects of the present invention include fixing intact tissues. The term "fixation" or "fixation" used herein refers to the process of preserving biological materials (e.g., tissues, cells, organelles, molecules, etc.) to avoid decay and / or degradation. Fixation can be completed according to any suitable scheme. Fixation can include contacting the sample with a fixing agent (i.e., a reagent containing at least one fixing agent). The sample can be contacted with the fixing agent for a long period of time, and the contact time specifically depends on the temperature, the nature of the sample, and the fixing agent. For example, the sample can be contacted with the fixing agent 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.

[0061] The sample can be contacted with the fixative agent for a period of time ranging from 5 minutes to 24 hours, for example, from 10 minutes to 20 hours, from 10 minutes to 18 hours, from 10 minutes to 12 hours, from 10 minutes to 8 hours, from 10 minutes to 6 hours, from 10 minutes to 4 hours, from 10 minutes to 2 hours, from 15 minutes to 20 hours, from 15 minutes to 18 hours, from 15 minutes to 12 hours, from 15 minutes to 8 hours, from 15 minutes to 6 hours, from 15 minutes to 4 hours, from 15 minutes to 2 hours, from 15 minutes to 1.5 hours, from 15 minutes to 1 hour, from 10 minutes to 30 minutes, from 15 minutes to 30 minutes, from 30 minutes to 2 hours, from 45 minutes to 1.5 hours, or from 55 minutes to 70 minutes.

[0062] Samples can be contacted with a fixing agent at various temperatures, depending on the protocol and reagents used. For example, in some cases, samples can be contacted with a fixing agent in a temperature range of -22°C to 55°C, wherein the temperature range of a particular purpose includes but is not limited to 50 to 54°C, 40 to 44°C, 35 to 39°C, 28 to 32°C, 20 to 26°C, 0 to 6°C, and -18 to -22°C. In some cases, samples can be contacted with a fixing agent at a temperature of -20°C, 4°C, room temperature (22-25°C), 30°C, 37°C, 42°C, or 52°C.

[0063] Any suitable fixing agent can be used. Common fixing agents include cross-linking fixing agents, precipitation fixing agents, oxidation fixing agents, mercury agents, etc. Cross-linking fixing agents chemically connect two or more molecules through covalent bonds, and various cross-linking agents can be used. Examples of suitable cross-linking fixing agents include, but are not limited to, aldehydes (e.g., formaldehyde, also commonly referred to as "paraformaldehyde" and "formalin"; glutaraldehyde, etc.), imidoesters, NHS (N-hydroxysuccinimide) esters, etc. Examples of suitable precipitation fixing agents include, but are not limited to, alcohols (e.g., methanol, ethanol, etc.), acetone, acetic acid, etc. In some embodiments, the fixing agent is formaldehyde (i.e., paraformaldehyde or formalin). The suitable final concentration of formaldehyde in the fixing agent is 0.1-10%, 1-8%, 1-4%, 1-2%, 3-5% or 3.5-4.5%, including about 1.6%, for 10 minutes. In some embodiments, the sample is fixed in formaldehyde at a final concentration of 4% (diluted with a more concentrated stock solution, e.g., 38%, 37%, 36%, 20%, 18%, 16%, 14%, 10%, 8%, 6%, etc.). In some embodiments, the sample is fixed in formaldehyde at a final concentration of 10%. In some embodiments, the sample is fixed in formaldehyde at a final concentration of 1%. In some embodiments, the fixative is glutaraldehyde. A suitable concentration of glutaraldehyde in the fixative is 0.1 to 1%. The fixative may contain more than one fixative in any combination. For example, in some embodiments, the sample is contacted with a fixative containing formaldehyde and glutaraldehyde.

[0064] The term "permeabilization" or "permeabilization" as used herein refers to the process of making cells (cell membranes, etc.) in a sample permeable to experimental reagents (e.g., nucleic acid probes, antibodies, chemical substrates, etc.). Any suitable method and / or reagent for permeabilization can be used. Suitable permeabilization reagents include detergents (e.g., saponin, Triton X-100, Tween-20, etc.), organic fixatives (e.g., acetone, methanol, ethanol, etc.), enzymes, etc. Detergents of various concentrations can be used. For example, permeabilization can be achieved using 0.001%-1% detergent, 0.05%-0.5% detergent, or 0.1%-0.3% detergent (e.g., 0.1% saponin, 0.2% tween-20, 0.1-0.3% triton X-100, etc.). In some embodiments, permeabilization is performed using methanol (on ice) for at least 10 minutes.

[0065] In some embodiments, the same solution is used as a fixative and a permeabilizing agent. For example, in some embodiments, the fixative contains 0.1%-10% formaldehyde and 0.001%-1% saponin. In some embodiments, the fixative contains 1% formaldehyde and 0.3% saponin.

[0066] The sample can be contacted with the permeabilizing agent for an extended period of time, depending on the temperature, the nature of the sample, and the permeabilizing agent. For example, the sample can be contacted with the permeabilizing agent 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 permeabilizing agent at a variety of temperatures, depending on the protocol and the agents used. For example, in some cases, the sample can be contacted with the permeabilizing agent at a temperature range of -82°C to 55°C, where specific temperature ranges of interest include, but are not limited to, 50 to 54°C, 40 to 44°C, 35 to 39°C, 28 to 32°C, 20 to 26°C, 0 to 6°C, -18 to -22°C, and -78 to -82°C. In some cases, the sample can be contacted with the permeabilizing agent at a temperature of -80°C, -20°C, 4°C, room temperature (22-25°C), 30°C, 37°C, 42°C, or 52°C.

[0067] In some embodiments, the sample is contacted with an enzymatic permeabilization reagent. The enzymatic permeabilization reagent permeates the sample by partially degrading the extracellular matrix or surface proteins (which hinder the determination reagent from penetrating the sample). After fixation, the enzymatic permeabilization reagent can be contacted at any time before target detection. In some cases, the enzymatic permeabilization reagent is proteinase K, a commercially available enzyme. In these cases, the sample is first contacted with proteinase K and then contacted with the fixation reagent. Proteinase K treatment (i.e., contact with proteinase K; also commonly referred to as "proteinase K digestion") can be performed for a period of time within a certain temperature range and within an enzyme concentration range that is empirically determined to be suitable for each cell type or tissue type being studied. 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 a concentration of 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 range of 2°C to 55°C, wherein specific temperature ranges of interest include, but are not limited to, 50 to 54°C, 40 to 44°C, 35 to 39°C, 28 to 32°C, 20 to 26°C, and 0 to 6°C. In some cases, 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 agent. In some embodiments, the sample is not contacted with proteinase K. The intact tissue is contacted with at least one fixative agent and one permeabilization agent to obtain a fixed permeabilized tissue. Ligase

[0068] In certain embodiments, the method disclosed herein includes adding a ligase to connect the second oligonucleotide and generate a closed nucleic acid ring. In certain embodiments, the adding of a ligase includes adding a DNA ligase. In an alternative embodiment, the second oligonucleotide is provided in the form of a closed nucleic acid ring, and the step of adding a ligase is omitted. In certain embodiments, a ligase is an enzyme that helps to sequence the target nucleic acid molecule.

[0069] The term "ligase" as used herein refers to an enzyme that is generally used to connect polynucleotides together or to connect the ends of a single polynucleotide. Ligase includes ATP-dependent double-stranded polynucleotide ligases, NAD-i-dependent double-stranded DNA or RNA ligases, and single-stranded polynucleotide ligases, for example, any of the ligases described in EC 6.5.1.1 (ATP-dependent ligase), EC 6.5.1.2 (NAD+-dependent ligase), EC 6.5.1.3 (RNA ligase). Specific examples of ligases include bacterial ligases, for example, Escherichia coli DNA ligase and Taq DNA ligase, Thermostable DNA ligase ( Technologies Corp. (Madison, Wisconsin), a subsidiary of ) and bacteriophage ligases, for example, T3 DNA ligase, T4 DNA ligase and T7 DNA ligase and mutants thereof. Rolling circle amplification

[0070] In some embodiments, the method of the present invention includes the step of performing rolling circle amplification in the presence of a nucleic acid molecule, wherein the operation includes using the second oligonucleotide as a template, and using the first oligonucleotide as a primer that prompts a polymerase to form one or more amplicons. In these embodiments, a single-stranded circular polynucleotide template is formed by connecting the second nucleotide, and the circular polynucleotide includes a region complementary to the first oligonucleotide. After adding a DNA polymerase in the presence of a suitable dNTP precursor and other cofactors, the first oligonucleotide is extended by replicating multiple template copies. After combining with a detection probe, the amplified product can be easily detected.

[0071] In some embodiments, only when the first oligonucleotide and the second oligonucleotide are hybridized with the same target nucleic acid molecule, the second oligonucleotide can be cyclized and subjected to rolling circle amplification to generate a cDNA nanosphere (i.e., an amplicon) containing multiple cDNA copies. The term "amplicon" refers to the amplified nucleic acid product of a PCR reaction or other nucleic acid amplification process. In some embodiments, amine-modified nucleotides are incorporated into the rolling circle amplification reactant.

[0072] Rolling circle amplification technology is well known in the art (see, e.g., Baner et al., Nucleic Acids Research, 26:5073-5078, 1998; Lizardi et al., Nature Genetics, 19:226, 1998; Schweitzer et al., Proc. Natl. Acad. Sci. USA, 97:101 13-1 19, 2000; Faruqi et al., BMC Genomics, 2:4, 2000; Nallur et al., Nucleic Acids Research, 29:el 18, 2001; Dean et al., Genome Research, 1 1:1095-1099, 2001; Schweitzer et al., Nature Biotechnology, 20:359-365, 2002; U.S. Pat. Nos. 6,054,274, 6,291,187, 6,323,009, 6,344,329, and 6,368,801). In some embodiments, the polymerase is phi29 DNA polymerase.

[0073] In some aspects, the nucleic acid molecule includes amine-modified nucleotides. In these embodiments, the amine-modified nucleotides include acrylic acid N-hydroxysuccinimide partial modification. Other examples of amine-modified nucleotides include but are not limited to 5-aminoallyl-dUTP partial modification, 5-propargylamino-dCTP partial modification, N6-6-aminohexyl-dATP partial modification or 7-deaza-7-propargylamino-dATP partial modification. Amplicon entrapment in tissue hydrogel environments

[0074] In some embodiments, the methods disclosed herein include embedding one or more amplicons in the presence of a hydrogel subunit to form one or more hydrogel-embedded amplicons. The hydrogel tissue chemistry methods described herein include covalently linking nucleic acids to in situ synthesized hydrogels for tissue clearing, enzyme diffusion, and multiple cycle sequencing, which are not possible with existing hydrogel tissue chemistry methods. In some embodiments, to embed the amplicons into a tissue hydrogel environment, amine-modified nucleotides are incorporated into a rolling circle amplification reaction, which is then functionalized with an acrylamide moiety (using acrylic acid N-hydroxysuccinimide ester) and copolymerized with acrylamide monomers to form a hydrogel.

[0075] As used herein, the term "hydrogel" or "hydrogel network" refers to a network formed by water-insoluble polymer chains, sometimes in the form of a colloidal gel, with water as the dispersion medium. In other words, a hydrogel is a class of polymeric materials that can absorb large amounts of water without dissolving. The water content of a hydrogel can exceed 99%, and the hydrogel can include natural or synthetic polymers or a combination thereof. In addition, due to its high water content, the flexibility of the hydrogel is very similar to that of natural tissue. Details of suitable hydrogels can be found in published U.S. Patent Application No. 20100055733, which is expressly incorporated herein by reference. As used herein, the term "hydrogel subunit" or "hydrogel precursor" refers to a hydrophilic monomer, prepolymer or polymer that can be cross-linked or "polymerized" to form a three-dimensional (3D) hydrogel network. Without being bound by any scientific theory, it is said that the fixation of the biological sample in the presence of the hydrogel subunit causes the components of the sample to cross-link with the hydrogel subunit, thereby fixing the molecular components in place while retaining the tissue structure and cell morphology.

[0076] In some embodiments, the embedding comprises copolymerizing the one or more amplicons with acrylamide. As used herein, the term "copolymer" refers to a polymer containing more than one subunit. The term encompasses polymers comprising two, three, four, five, or six subunits.

[0077] In certain aspects, the embedding comprises making the one or more hydrogel-embedded amplicons transparent, wherein the target nucleic acid is substantially retained in the one or more hydrogel-embedded amplicons. In these embodiments, the transparentization comprises substantially removing a variety of cellular components in the one or more hydrogel-embedded amplicons. In some other embodiments, the transparentization comprises substantially removing lipids in the one or more hydrogel-embedded amplicons. The term "substantially" as used herein refers to a reduction of about 70% or more in the initial amount present in the sample before transparentization, for example, a reduction of 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, and 100%.

[0078] In some embodiments, making the hydrogel-embedded amplicon transparent comprises subjecting the sample to electrophoresis. In some embodiments, the amplicon is subjected to electrophoresis using a buffer solution comprising an ionic surfactant. In some embodiments, the ionic surfactant is sodium dodecyl sulfate (SDS). In some embodiments, the sample is subjected to electrophoresis using a voltage of about 10 to about 60 volts. In some embodiments, the sample is subjected to electrophoresis for a period of time ranging from about 15 minutes to about 10 days. In some embodiments, the method further involves incubating the transparentized sample in a mounting medium having a refractive index that matches the refractive index of the transparentized tissue. In some embodiments, the mounting medium increases the light transmittance of the sample. In some embodiments, the mounting medium comprises glycerol. Sequencing Error Correction by Dynamic Annealing and Ligation (SEDAL)

[0079] The method disclosed herein includes a step of contacting the one or more hydrogel-embedded amplicons with a barcode sequence with a pair of primers under conditions that allow connection, wherein the primer pair includes a third oligonucleotide and a fourth oligonucleotide, wherein the connection is achieved only when the third oligonucleotide and the fourth oligonucleotide are connected to the same amplicon. In some embodiments, SEDAL is designed for STARmap. In these embodiments, the improved connection sequencing method described herein includes operating at room temperature to maintain optimal tissue morphology with low background noise and reduced error rate. In these other embodiments, contacting the one or more hydrogel-embedded amplicons includes eliminating error accumulation during sequencing.

[0080] In some embodiments, contacting the one or more hydrogel-embedded amplicons occurs two or more times, including but not limited to, for example, three or more times, four or more times, five or more times, six or more times, or seven or more times. In certain embodiments, for thin tissue samples, contacting the one or more hydrogel-embedded amplicons occurs four or more times. In other embodiments, for thick tissue samples, contacting the one or more hydrogel-embedded amplicons occurs six or more times. In some embodiments, one or more amplicons can be contacted with a pair of primers 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.

[0081] The sample prepared by the target method can be analyzed by any of a variety of different microscopic techniques, such as optical microscopy (e.g., bright field, oblique illumination, dark field, phase contrast, differential interference contrast, interference reflection, epifluorescence, confocal, etc.), laser microscopy, electron microscopy, and scanning probe microscopy. In some aspects, the non-transitory computer-readable medium first converts the raw images of multiple rounds of in situ sequencing obtained by microscopy into decoded gene identities and spatial locations, and then analyzes the gene expression composition of each cell. SEDAL oligonucleotide primers

[0082] In certain embodiments, the method disclosed herein includes a third oligonucleotide and a fourth oligonucleotide. In some aspects, the third oligonucleotide is configured to decode bases, and the fourth oligonucleotide is configured to convert decoded bases into signals. In some aspects, the signal is a fluorescent signal. In exemplary aspects, under conditions allowing connection, the one or more hydrogel-embedded amplicons with a barcode sequence are contacted with a pair of primers, and it is related to make the third oligonucleotide and the fourth oligonucleotide each be connected to form a stable product, so that imaging operations are performed only in the case of perfect matching. In some aspects, the mismatch sensitivity of the ligase is used to determine the basic sequence of the target nucleic acid molecule.

[0083] In the case of duplexes, the term "perfect match" refers to the polynucleotide and / or oligonucleotide chains that constitute the duplex being bound to each other to form a double-stranded structure such that each nucleotide in each chain is Watson-Crick base paired with a nucleotide in the other chain. The term "duplex" includes, but is not limited to, the pairing of nucleoside analogs that can be used, for example, deoxyinosine, nucleosides with 2-aminopurine bases, peptide nucleic acids (PNA), etc. A "mismatch" in a duplex between two oligonucleotides refers to a pair of nucleotides in the duplex that fail to achieve Watson-Crick bonding.

[0084] In some embodiments, the method comprises a plurality of third oligonucleotides hybridized to the target nucleotide sequence, including but not limited to 5 or more third oligonucleotides, for example, 8 or more, 10 or more, 12 or more, 15 or more, 18 or more, 20 or more, 25 or more, 30 or more, 35 or more. In some embodiments, the method of the present invention comprises a plurality of third oligonucleotides, including but not limited to 15 or more third oligonucleotides, for example, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more and up to 80 different first oligonucleotides, which hybridize to 15 or more, for example, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more and up to 80 different target nucleotide sequences. In certain embodiments, the method includes multiple fourth oligonucleotides, including but not limited to 5 or more fourth oligonucleotides, for example, 8 or more, 10 or more, 12 or more, 15 or more, 18 or more, 20 or more, 25 or more, 30 or more, 35 or more. In certain embodiments, the method of the present invention includes multiple fourth oligonucleotides, including but not limited to 15 or more fourth oligonucleotides, for example, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more and 80 different first oligonucleotides at the most, which are hybridized with 15 or more, for example, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more and 80 different target nucleotide sequences at the most. Multiple oligonucleotide pairs can be used in the reaction, wherein one or more oligonucleotide pairs are specifically bound to each target nucleic acid. For example, two primer pairs can be used for a target nucleic acid to improve sensitivity and reduce variability. It is also contemplated to detect multiple different target nucleic acids in a cell, for example, detecting 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.

[0085] In certain embodiments, SEDAL relates to a ligase whose activity is hindered by a base mismatch, a third oligonucleotide, and a fourth oligonucleotide. As used herein, the term "hindered" refers to a decrease in the activity of the ligase by about 20% or more, for example, by 25% or more, 50% or more, 75% or more, 90% or more, 95% or more, 99% or more, and 100%. In some embodiments, the length of the third oligonucleotide is 5-15 nucleotides, including but not limited to 5-13 nucleotides, 5-10 nucleotides, or 5-8 nucleotides. In some embodiments, the T of the third oligonucleotide is m is room temperature (22-25°C). In some embodiments, the third oligonucleotide is a degenerate oligonucleotide or a portion thereof. In some embodiments, the length of the fourth oligonucleotide is 5-15 nucleotides, including but not limited to 5-13 nucleotides, 5-10 nucleotides, or 5-8 nucleotides. In some embodiments, the T of the fourth oligonucleotide is m The fourth oligonucleotide can be stripped after each SEDAL cycle corresponding to a base read, thereby eliminating error accumulation during sequencing. In these embodiments, the fourth oligonucleotide is stripped with formamide.

[0086] In some embodiments, SEDAL involves washing of the third and fourth oligonucleotides to remove unbound oligonucleotides, and then revealing fluorescent products for imaging. In certain exemplary embodiments, detectable labels can be used to detect one or more nucleotides and / or oligonucleotides described herein. In certain embodiments, detectable labels can be used to detect the one or more amplicons. Examples of detectable labels include various radioactive moieties, enzymes, prosthetic groups, fluorescent labels, luminescent labels, bioluminescent labels, 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, dichlorotriazine aminofluorescein, dansyl chloride, phycoerythrin, etc. Examples of bioluminescent labels include, but are not limited to, luciferase (e.g., bacteria, fireflies, click beetles, etc.), luciferin, aequorin, etc. Examples of enzyme systems with visually detectable signals include, but are not limited to, beta-galactosidase, glucuronidase, phosphatase, peroxidase, cholinesterase, etc. Identifiable labels also include radioactive compounds, e.g. 125 I. 35 S. 14 C or 3 H. Identifiable markers are commercially available from a variety of sources.

[0087] Fluorescent labels and their attachment to nucleotides and / or oligonucleotides are described in detail in a number of reviews, including: Haugland, Handbook of Fluorescent Probes and Research Chemicals, 9th Edition (Molecular Probes, Inc., Eugene, 2002); Keller and Manak, DNA Probes, 2nd Edition (Stockton Press, New York, 1993); Eckstein, ed., Oligonucleotides and Analogs: A Practical Approach (IRL Press, Oxford, 1991); and Wetmur, Reviews in Biochemistry and Molecular Biology, 26:227-259 (1991). Specific methods suitable for use in the present invention are described in the following references: U.S. Pat. Nos. 4,757,141, 5,151,507, and 5,091,519. In one aspect, one or more fluorescent dyes are used as labels for the labeled target sequence, for example, as described in U.S. Pat. No. 5,188,934 (4,7-dichlorofluorescein dye); U.S. Pat. No. 5,366,860 (spectrally resolvable rhodamine dye); U.S. Pat. No. 5,847,162 (4,7-dichlororhodamine dye); U.S. Pat. No. 4,318,846 (ether-substituted fluorescein dye); U.S. Pat. No. 5,800,996 (energy transfer dye); Lee et al.; U.S. Pat. No. 5,066,580 (xanthine dye); U.S. Pat. No. 5,688,648 (energy transfer dye), etc. Labeling can also be done with quantum dots, as described in the following patents and patent publications: 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 U.S. Patents. As used herein, the term "fluorescent label" includes a signaling moiety that transmits information through the fluorescence absorption and / or emission characteristics of one or more molecules. The fluorescence characteristics include fluorescence intensity, fluorescence lifetime, emission spectrum characteristics, energy transfer, etc.

[0088] Commercially available fluorescent nucleotide analogs that are readily incorporated into nucleotide and / or oligonucleotide sequences include, but are not limited to, Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dUTP (Amersham Biosciences, Piscataway, NJ), fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, TEXASRED TM -5-dUTP, CASCADE BLUE TM-7-dUTP, BODIPY TMFL-14-dUTP, BODIPY TMR-14-dUTP, BODIPY TMTR-14-dUTP, RHODAMINEGREEN TM -5-dUTP、OREGON GREENR TM 488-5-dUTP, TEXAS RED TM -12-dUTP、BODIPY TM 630 / 650-14-dUTP, BODIPY TM 650 / 665-14-dUTP、ALEXA FLUOR TM 488-5-dUTP、ALEXA FLUOR TM 532-5-dUTP、ALEXAFLUOR TM 568-5-dUTP、ALEXA FLUOR TM 594-5-dUTP、ALEXA FLUOR TM 546-14-dUTP, fluorescein-12-UTP, tetramethylrhodamine-6-UTP, TEXAS RED TM -5-UTP, mCherry, CASCADEBLUE TM -7-UTP, BODIPY TM FL-14-UTP, BODIPY TMR-14-UTP, BODIPY TM TR-14-UTP、RHODAMINE GREEN TM -5-UTP、ALEXA FLUOR TM 488-5-UTP, LEXA FLUOR TM 546-14-UTP (Molecular Probes, Inc. Eugene, OR), etc. Protocols for custom synthesis of nucleotides with other fluorophores are well known in the art (see, Henegariu et al. (2000), Nature Biotechnology, 18:345).

[0089] Other fluorophores that can be used for post-synthesis attachment include, but are not limited to, ALEXA FLUOR TM 350、ALEXAFLUOR TM 532. ALEXA FLUOR TM 546. ALEXA FLUOR TM 568、ALEXAFLUOR TM 594、ALEXAFLUOR TM647, 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 Biology Probes, Inc. (Eugene, OR)), Cy2, Cy3.5, Cy5.5, Cy7 (Amersham Biosciences, Piscataway, NJ), etc. FRET tandem fluorophores can also be used, including but not limited to PerCP-Cy5.5, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, APC-Cy7, PE-Alexa dyes (610, 647, 680), APC-Alexa dyes, etc.

[0090] Metallic silver or gold particles can be used to enhance the signal from fluorescently labeled nucleotide and / or oligonucleotide sequences (Lakowicz et al. (2003), Biotechnology, 34:62).

[0091] Biotin or its derivatives can also be used as labels on nucleotide and / or oligonucleotide sequences and subsequently bound to detectably labeled avidin / streptavidin derivatives (e.g., phycoerythrin-conjugated streptavidin) or detectably labeled anti-biotin antibodies. Digoxigenin can be incorporated as a label and subsequently bound to detectably labeled anti-digoxigenin antibodies (e.g., fluoresceinized anti-digoxigenin). Aminoallyl-dUTP residues can be incorporated into oligonucleotide sequences and subsequently coupled to fluorescent dyes derived from N-hydroxysuccinimide (NHS). In general, any member of a conjugate pair can be incorporated into a detection oligonucleotide, provided that a detectably labeled conjugated partner can be bound for detection. The term "antibody" as used herein refers to any type of antibody molecule or any subfragment thereof, e.g., Fab.

[0092] Other suitable labels for oligonucleotide sequences may include fluorescein (FAM), digoxigenin, dinitrophenol (DNP), dansyl, biotin, bromodeoxyuridine (BrdU), hexahistidine (6×His), phosphoamino acids (e.g., P-tyr, P-ser, P-thr), etc. In one embodiment, the following hapten / antibody pairs are used in the detection, wherein each antibody is derivatized with a detectable label: biotin / α-biotin, digoxigenin / α-digoxigenin, dinitrophenol (DNP) / α-DNP, 5-carboxyfluorescein (FAM) / α-FAM.

[0093] In certain exemplary embodiments, the nucleotide and / or oligonucleotide sequence may be indirectly labeled, particularly with a hapten, and then bound to a capture agent, for example, as described in U.S. Pat. Nos. 5,344,757, 5,702,888, 5,354,657, 5,198,537, and 4,849,336, PCT Publication No. WO 91 / 17160, and the like. There are many different hapten-capture agent pairs available for use. Exemplary haptens include, but are not limited to, biotin, debiotin and other derivatives, dinitrophenol, dansyl, fluorescein, CY5, digoxigenin, and the like. For biotin, the capture agent may be avidin, streptavidin, or an antibody. Antibodies may be used as capture agents for other haptens (many dye-antibody pairs are commercially available, for example, Molecular Probes, Eugene, Oregon). cell

[0094] The methods disclosed herein include a method for in situ gene sequencing of target nucleic acids in cells within intact tissues. In certain embodiments, the cells are present in a cell population. In certain other embodiments, the cell population includes a variety of cell types, including but not limited to excitatory neurons, inhibitory neurons, and non-neuronal cells. The cells used in the assay of the present invention can be an organism, a single cell type derived from an organism, or a mixture of various cell types. The present invention includes naturally occurring cells and cell populations, genetically modified cell lines, cells derived from transgenic animals, and the like. Almost all cell types and sizes can be included. Suitable cells include bacteria, fungi, plants, and animal cells. In one embodiment of the present invention, the cells are mammalian cells, for example, complex cell populations, such as naturally occurring tissues, for example, blood, liver, pancreas, neural tissue, bone marrow, skin, and the like. Some tissues may be dissociated to make monodisperse suspensions. Alternatively, the cells can be cultured cell populations, for example, cultures derived from complex cell populations, cultures derived from single cell types, wherein the cells have differentiated into multiple lineages, or wherein the cells respond differently to stimuli, and the like.

[0095] The cell types used in the present invention include stem cells (e.g., embryonic stem cells, hematopoietic stem cells, mesenchymal stem cells, neural crest cells, etc.), endothelial cells, muscle cells, cardiomyocytes, smooth muscle cells and skeletal muscle cells, mesenchymal cells, epithelial cells; hematopoietic cells, for example, lymphocytes, including T cells, such as Th1 T cells, Th2 T cells, ThO T cells, cytotoxic T cells; B cells, pre-B cells, etc.; monocytes; dendritic cells; neutrophils; macrophages; natural killer cells; mast cells, etc.; adipocytes, cells associated with specific organs, such as thymus, endocrine glands, pancreas, brain, such as neurons, glial cells, astrocytes, dendritic cells, etc., and genetically modified cells thereof. Hematopoietic cells may be associated with inflammatory processes, autoimmune diseases, etc., endothelial cells, smooth muscle cells, cardiomyocytes, etc. may be associated with cardiovascular diseases; almost all types of cells may be associated with neoplasia, such as sarcomas, cancers, and lymphomas; liver diseases associated with hepatocytes; kidney diseases associated with kidney cells; and so on.

[0096] The cells may also be different types of transformed or neoplastic cells, cancers derived from different cell sources, lymphomas derived from different cell types, etc. The American Type Culture Collection (ATCC) (Manassas, Virginia) has collected and provided more than 4,000 cell lines from more than 150 different species, more than 950 cancer cell lines, including 700 human cancer cell lines. The National Cancer Institute (NCI) has compiled clinical, biochemical and molecular data on a large number of human tumor cell lines, which can be obtained from ATCC or NCI (Phelps et al. (1996), Journal of Cell Biochemistry Supplement, 24: 32-91). The present invention includes different cell lines derived spontaneously, or cell lines selected for the growth or response characteristics required of a single cell line; and may include multiple cell lines derived from similar tumor types but from different patients or sites.

[0097] The cell can be a non-adherent cell (e.g., a blood cell, including a monocyte, a T cell, a B cell; a tumor cell, etc.) or an adherent cell (e.g., an epithelial cell, an endothelial cell, a neural cell, etc.). In order to obtain the expression profile of the adherent cell, the adherent cell is separated from the matrix and other cells to which it is adhered, while maintaining its ability to recognize and bind to the probe molecule.

[0098] The cells can be obtained from various tissues (e.g., blood, bone marrow), solid tissues (e.g., solid tumors), and ascites of an individual using various techniques known in the art, such as extraction, lavage, washing, and surgical dissection. Cells can be obtained from fixed or unfixed, fresh or frozen, intact or dissociated samples. Tissues can be mechanically or enzymatically dissociated using known techniques. Imaging

[0099] The methods disclosed herein include imaging the one or more hydrogel-embedded amplicons using any of a variety of different types of microscopy techniques, such as confocal microscopy, two-photon microscopy, light-field microscopy, whole tissue expansion microscopy, and / or CLARITY TM Optimized monolayer light microscopy (COLM).

[0100] The simplest of all optical microscopy techniques is brightfield microscopy. The sample is illuminated by transmitted white light, i.e., illuminated from below and observed from above. Limitations of this microscopy technique include low contrast in most biological samples and low apparent resolution due to loss of focus of in-focus material. Significant advantages of this technique are its simplicity and minimal sample preparation.

[0101] With oblique illumination microscopy, the specimen is illuminated from the side. This gives the image a three-dimensional appearance and can highlight otherwise invisible features. The latest technology based on this approach is Hoffman modulation contrast microscopy, a system used for cell cultures on an inverted microscope. While oblique illumination suffers from the same limitations as brightfield microscopy (low contrast in most biological specimens; lower apparent resolution due to out-of-focus objects), it can highlight otherwise invisible structures.

[0102] Darkfield microscopy is a technique used to improve contrast in unstained, transparent specimens. Darkfield illumination uses a carefully aligned light source to minimize the amount of directly transmitted (unscattered) light entering the image plane, while collecting only light scattered by the specimen. Darkfield can significantly improve image contrast (especially for transparent objects) without requiring any equipment setup or specimen preparation. However, the final image of many biological specimens using this technique has lower light intensity and the apparent resolution remains low.

[0103] Phase contrast is an optical microscopy illumination technique that converts the phase shift of light passing through a transparent specimen into brightness variations in the image. In other words, phase contrast represents differences in refractive index as contrast differences. The phase shift itself is invisible to the human eye, but becomes visible when it is represented as brightness variations.

[0104] In differential interference contrast (DIC) microscopy, differences in light density appear as differences in relief. The system consists of special prisms (Nomarski, Wollaston) in the condenser that separate the light into an ordinary and an extraordinary beam. The spatial difference between the two beams is small (less than the maximum resolution of the objective). After the beams have passed through the specimen, they are reunited in the objective by similar prisms. In a homogeneous specimen, there is no difference between the two beams and no contrast is produced. However, near refractive boundaries (e.g., the nucleus within the cytoplasm), the difference between the ordinary and extraordinary beams can produce a relief effect in the image. Differential interference contrast requires a polarized light source to work; two polarizing filters must be installed in the light path, one below the condenser (polarizer) and the other above the objective (analyzer).

[0105] Another interference microscopy technique is interference reflection microscopy (also known as reflection interference contrast or RIC). This microscopy technique is used to detect cell adhesion to a glass surface when there is an interface between two materials with different refractive indices, using polarized light to be reflected in a narrow range of wavelengths. Whenever cells are attached to a glass surface, interference occurs between the reflected light from the glass and the reflected light from the attached cells. If there are no cells attached to the glass, no interference occurs.

[0106] Fluorescence microscopy is a type of optical microscopy that uses fluorescence and phosphorescence instead of, or in addition to, reflection and absorption to study the properties of organic or inorganic matter. In fluorescence microscopy, a sample is illuminated with light of a wavelength that excites it to produce fluorescence. The fluorescence, which is usually of a longer wavelength than the illumination, is then imaged through the microscope objective. The technique can use two filters; an illumination (or excitation) filter that ensures that the illumination is nearly monochromatic and of the appropriate wavelength, and a second emission (or blocking) filter that ensures that no light from the excitation source reaches the detector. Alternatively, both of these functions can be accomplished with a single dichroic filter. The term "fluorescence microscope" refers to any microscope that uses fluorescence to produce images, whether it is a simpler setup such as an epifluorescence microscope or a more complex design such as a confocal microscope that can use optical sectioning to obtain fluorescence images with higher resolution.

[0107] Confocal microscopy uses point illumination and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus signals. Because only light from fluorescence very close to the focal plane is detected, the optical resolution of the image, especially along the depth of the specimen, far exceeds that of widefield microscopy. However, this increase in resolution comes at the expense of reduced signal intensity—often requiring long exposures—because much of the light from specimen fluorescence is blocked by the pinhole. Because only one point in the specimen is illuminated at a time, 2D or 3D imaging requires scanning across the specimen in a regular raster (i.e., a rectangular pattern of parallel scan lines). The achievable thickness of the focal plane depends primarily on the wavelength of the light used divided by the numerical aperture of the objective, and also on the optical properties of the specimen. Thin optical sections may make these types of microscopes particularly good at 3D imaging and profiling of specimen surfaces. COLM is another microscopy technique that allows for rapid 3D imaging of large clarified specimens. COLM interrogates large immunohistochemically stained tissues, allowing for increased acquisition speeds and, therefore, the quality of the data generated.

[0108] With single plane illumination microscopy (SPIM), also known as single-sheet light microscopy, only fluorophores in the focal plane of the detection objective are illuminated. The single-sheet light is a beam of light that is collimated in one direction and focused in another. The method also provides inherent optical sectioning, since no fluorophores are excited outside the focal plane of the detector. In addition, single-sheet light methods reduce photobleaching, phototoxicity, and typically allow for more scans per specimen than conventional microscopy techniques. By rotating the specimen, the technique can image nearly any plane using multiple views obtained from different angles. However, only relatively thin portions of the specimen are imaged with high resolution at each angle, and the image becomes increasingly blurred as the thickness increases.

[0109] Super-resolution microscopy is an optical microscopy technique. The resolution of conventional optical microscopy is limited by the diffraction of light, as demonstrated by Ernst Abbe in 1873. The FWHM (full width at half maximum) value of the point spread function is a good approximation of the achievable resolution, which is typically around 250nm for sophisticated wide-field microscopes with high numerical aperture and for visible light. Super-resolution techniques allow images to be captured with resolutions above the diffraction limit. The techniques can be divided into two main categories: "practical" super-resolution techniques, which capture the information contained in the evanescent waves, and "functional" super-resolution techniques, which use experimental techniques and known limitations of the imaged object to reconstruct a super-resolution image.

[0110] Laser microscopy employs laser illumination sources used in a variety of microscopy techniques. For example, laser microscopy focused on biological applications employs ultrashort pulse lasers or femtosecond lasers used in a variety of techniques including nonlinear microscopy, saturation microscopy, and multiphoton fluorescence microscopy, such as two-photon excitation microscopy (a fluorescence imaging technique that can image thick living tissues, for example, up to a millimeter thick).

[0111] In electron microscopy (EM), a beam of electrons is used to illuminate a specimen and produce a magnified image. Electron microscopy has higher resolving power than photodynamic optical microscopy because the wavelength of electrons is about 100,000 times shorter than visible light (photons). Electron microscopy can achieve resolutions of better than 50 pm and magnifications up to about 10,000,000 times, whereas ordinary non-confocal optical microscopy is diffraction-limited, with a resolution of about 200 nm and effective magnifications of less than 2000 times. Electron microscopy uses 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, which form a magnified image by focusing light on or passing it through the specimen. Electron microscopes are used to observe a variety of biological and inorganic specimens, including microorganisms, cells, macromolecules, biopsies, metals, and crystals. In industry, electron microscopes are often used for quality control and failure analysis. Examples of electron microscopy include transmission electron microscopy (TEM), scanning electron microscopy (SEM), reflection electron microscopy (REM), scanning transmission electron microscopy (STEM), and low voltage electron microscopy (LVEM).

[0112] Scanning probe microscopy (SPM) is a microscopy technique that uses a physical probe that is scanned over a specimen to form an image of a surface. The surface image can be obtained by mechanically moving the probe line by line over a raster scanned area of ​​the specimen and recording how the probe-surface interaction changes with position. Examples of SPM include atomic force microscopy (ATM), ballistic electron emission microscopy (BEEM), chemical force microscopy (CFM), conductive atomic force microscopy (C-AFM), electrochemical scanning tunneling microscopy (ECSTM), electrostatic force microscopy (EFM), fluid force microscopy (FluidFM), force modulation microscopy (FMM), feature-oriented scanning probe microscopy (FOSPM), Kelvin probe force microscopy (KPFM), magnetic force microscopy (MFM), magnetic resonance force microscopy (MRFM), near-field scanning optical microscopy (NSOM) (or scanning near-field optical microscopy (SNOM)), piezoelectric response force microscopy (Piezoelectric force microscopy). technology (PFM), photon scanning tunneling microscopy (PSTM), photothermal microspectroscopy / microscopy (PTMS), scanning capacitance microscopy (SCM), scanning electrochemical microscopy (SECM), scanning gated microscopy (SGM), scanning Hall probe microscopy (SHPM), scanning ion conductivity microscopy (SICM), spin-polarized scanning tunneling microscopy (SPSM), scanning spreading resistance microscopy (SSRM), scanning thermal microscopy (SThM), scanning tunneling microscopy (STM), scanning tunneling potentiometry (STP), scanning voltage microscopy (SVM) and synchrotron X-ray scanning tunneling microscopy (SXSTM).

[0113] Whole tissue expansion microscopy (exM) allows imaging of thick preserved specimens with a lateral resolution of approximately 70 nm. With ExM, the optical diffraction limit can be circumvented by physically swelling the biological specimen prior to imaging, bringing sub-diffraction-limited structures into the size range observable with conventional diffraction-limited microscopy. ExM can image biological specimens at the voxel rate of a diffraction-limited microscope, but at the voxel size of a super-resolution microscope. Swelled samples are transparent and index-matched to water, as the water content of the swelled material is >99%. Expansion microscopy is well known in the art, for example, as described in the following publication: Gao et al., Q&A: Expansion microscopy, BMC Biol, 2017;15:50. Screening methods

[0114] The method disclosed herein also provides a method for screening a candidate agent to determine whether the candidate agent can regulate gene expression of a nucleic acid in a cell in an intact tissue, the method comprising: (a) contacting a fixed permeabilized intact tissue with at least one pair of oligonucleotide primers under conditions that allow specific hybridization to occur, wherein the primer pair comprises a first oligonucleotide and a second oligonucleotide; wherein the first oligonucleotide and the second oligonucleotide each comprise a first complementary region, a second complementary region, and a third complementary region; wherein the second oligonucleotide further comprises a barcode sequence; wherein the first complementary region of the first oligonucleotide is complementary to a first portion of the target nucleic acid, wherein the second complementary region of the first oligonucleotide is complementary to the first complementary region of the second oligonucleotide, wherein the third complementary region of the first oligonucleotide is complementary to the third complementary region of the second oligonucleotide, wherein the second complementary region of the second oligonucleotide is complementary to a second portion of the target nucleic acid, and wherein the first complementary region of the first oligonucleotide is adjacent to the second complementary region of the second oligonucleotide; (b) adding a ligase to connect the second oligonucleotide and form a closed nucleic acid circle; (c) (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, wherein the primer pair includes a third oligonucleotide and a fourth oligonucleotide, wherein ligation is achieved only when the third oligonucleotide and the fourth oligonucleotide are ligated to the same amplicon; (f) repeating step (e); (g) imaging the one or more hydrogel-embedded amplicons to determine in situ gene sequencing of target nucleic acids in cells within intact tissues; and (h) detecting gene expression levels of the target nucleic acids, wherein a change in the expression level of the target nucleic acids in the presence of the at least one candidate agent relative to the expression level of the target nucleic acids in the absence of the at least one candidate agent indicates that the at least one candidate agent regulates gene expression of nucleic acids in cells within intact tissues. The screening method includes the STARmap steps provided herein.

[0115] In some aspects, the detection includes the implementation of flow cytometry; sequencing; probe binding and electrochemical detection; pH change; enzyme-induced catalysis combined with DNA tags; quantum entanglement; Raman spectroscopy; terahertz wave technology; and / or scanning electron microscopy. In some aspects, the flow cytometry is a large number of cell counting methods or fluorescence activated flow cytometry. In some other aspects, the detection includes the implementation of microscopy, scanning mass spectrometry, or other imaging techniques described herein. In these aspects, the detection includes determining a signal, for example, a fluorescent signal.

[0116] As used herein, the terms "test agent," "candidate agent," and grammatically equivalent expressions are used interchangeably and refer to any molecule (e.g., protein (including proteins, polypeptides, and peptides herein), small (i.e., 5-1000 Da, 100-750 Da, 200-500 Da, or less than 500 Da in size) molecule, organic or inorganic molecule, polysaccharide, polynucleotide, etc.) whose activity is to be determined in a subject assay.

[0117] Various candidate agents can be screened out by the above method.Candidate agents cover a variety of chemical classifications, for example, molecular weight exceeds 50 Daltons (for example, at least about 50Da, at least about 100Da, at least about 150Da, at least about 200Da, at least about 250Da or at least about 500Da) and molecular weight is 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 of small size organic compounds. For example, in some embodiments, suitable candidate agents are organic compounds with a molecular weight range of about 500Da to about 20,000Da, for example, about 500Da to about 1000Da, about 1,000Da to about 2,000Da, about 2,000Da to about 2,500Da, about 2,500Da to about 5,000Da, about 5,000Da to about 10,000Da or about 10,000Da to about 20,000Da.

[0118] Candidate agents can include functional groups necessary for structural interaction (e.g., hydrogen bonding) with proteins, and can include at least one amino, carbonyl, hydroxyl or carboxyl group, or at least two functional chemical groups. Candidate agents can include cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above-mentioned functional groups. Candidate agents can also be derived from biomolecules including peptides, carbohydrates, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

[0119] Candidate agents can be obtained from various sources, including synthetic or natural compound libraries. For example, many means can be used for the random and directed synthesis of various organic compounds and biomolecules, including the expression of random oligonucleotides and oligopeptides. Alternatively, natural compound libraries in the form of bacterial, fungal, plant and animal extracts can be obtained or easily produced. In addition, natural or synthetically produced libraries and compounds can be easily modified by conventional chemical, physical and biochemical means, and can be used to produce combinatorial libraries. Known agents can be directly or randomly chemically modified (e.g., acylated, alkylated, esterified, amidated, etc.) to generate structural analogs. In addition, screening can be performed for known pharmacologically active compounds and chemical analogs thereof, or for new agents with unknown properties, such as agents produced by rational drug design.

[0120] In one embodiment, the candidate modulator is a synthetic compound. Many techniques are available for random and directed synthesis of various organic compounds and biomolecules, including expression of random oligonucleotides. See, for example, WO 94 / 24314, which is expressly incorporated herein by reference, which discusses methods for generating new compounds, including random chemical methods and enzymatic methods.

[0121] In another embodiment, the candidate agents are provided as libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts that are available or readily produced. In addition, natural or synthetically produced libraries and compounds can be easily modified by conventional chemical, physical and biochemical means. Known agents can be chemically modified (including enzymatically) directly or randomly to generate structural analogs.

[0122] In one embodiment, candidate agents include proteins (including 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 the protein can be tested, or random or directed digests of cell extracts containing the protein can be tested. Prokaryotic and eukaryotic protein libraries can be prepared for screening in this manner. Other embodiments include bacterial, fungal, viral, and mammalian protein (e.g., human protein) libraries.

[0123] In one embodiment, the candidate agent is an organic moiety. In this embodiment, as generally described in WO 94 / 243 14, the candidate agent is synthesized from a series of substrates that can be chemically modified. "Chemical modification" as described herein includes traditional chemical reactions and enzymatic reactions. These substrates generally include, but are not limited to, alkyl groups (including alkanes, alkenes, alkynes and heteroalkyls), aryl groups (including aromatics and heteroaryls), alcohols, ethers, amines, aldehydes, ketones, acids, esters, amides, cyclic compounds, heterocyclic compounds (including purines, pyrimidines, benzodiazepines, β-lactams, tetracyclines, cephalosporins and carbohydrates), steroids (including estrogens, androgens, cortisone, ecdysone, etc.), alkaloids (including ergot, periwinkle, curare, pyrrolidine and mitomycin), organometallic compounds, compounds containing heteroatoms, amino acids and nucleosides. The moiety can be chemically (including enzymatically) reacted to form a new substrate or candidate agent, which can then be tested using the present invention. Equipment and systems

[0124] The present invention also includes devices for implementing various aspects of the subject methods. The subject devices may include, for example, imaging chambers, electrophoresis devices, flow chambers, microscopes, needles, tubes, pumps.

[0125] The present invention also provides a system for implementing the target method. The system may include one or more of the modules described herein. For example, a power supply, a refrigeration unit, a waste disposal, a heating unit, a pump, etc. The system may also include any reagent described herein, for example, an imaging buffer, a cleaning buffer, a stripping buffer, Nissl and DAPI solutions. The system according to certain embodiments may also include a microscope and / or related imaging equipment, for example, a camera assembly, a digital imaging assembly and / or an image capture device, a computer processor configured to collect images according to one or more user inputs, etc.

[0126] As described above, the system described herein includes a fluidic device having an imaging chamber and a pump; and a processor unit configured to implement a method for in situ gene sequencing of a target nucleic acid in a cell in an intact tissue as described herein. In some embodiments, the system is capable of automating STARmap, a process described herein as including but not limited to: repeatedly hybridizing the probes with DNA embedded in a gel; connecting fluorescently labeled oligonucleotides to these probes; washing away excess probes; imaging; and stripping the probes for the next round of sequencing. In some embodiments, the system can operate continuously. In some embodiments, the system includes an imaging chamber for flowing sequencing chemicals involved in the in situ DNA sequencing process through the sample. In some embodiments, the fluid and pump system controls the sequencing chemicals delivered to the sample.

[0127] Buffer can be added / removed / recirculated / replaced using one or more ports, and optionally using tubing, pumps, valves, or any other suitable fluid handling and / or fluid handling equipment (e.g., tubing removably attached or permanently attached to one or more components of the device). For example, a first tubing having a first end and a second end can be attached to a first port, and a second tubing having a first end and a second end can be attached to a second port, wherein the first end of the first tubing is attached to the first port, and the second end of the first tubing is operably connected to a container, such as a cooling unit, a heating unit, a filtration unit, a waste container, etc.; and the first end of the second tubing is attached to the second port, and the second end of the second tubing is operably connected to a container, such as a cooling unit, a beaker placed on ice, a filtration unit, a waste container, etc.

[0128] In some embodiments, the system includes a non-transitory computer-readable storage medium having instructions that, when executed by the processor unit, cause the processor unit to control the delivery of chemicals and synchronize the process with microscope-related operations. In some embodiments, the non-transitory computer-readable storage medium includes instructions that, when executed by the processor unit, cause the processor unit to measure light signals. utility

[0129] The apparatus, methods and systems of the present invention have many uses in the art, for example, for biomedical research and / or clinical diagnosis. For example, in biomedical research, applications include, but are not limited to, spatially resolved gene expression analysis for basic biology or drug screening. In clinical diagnosis, applications include, but are not limited to, detecting gene markers of patient samples, for example, diseases, immune responses, bacterial or viral DNA / RNA. Examples of the advantages of the methods described herein include efficiency, where it takes only 3 or 4 days to obtain final data from the original sample, and the data is provided much faster than the corresponding speed of existing microarray or sequencing technologies; high multiplexity (up to 1000 genes); single cell and single molecule sensitivity; preservation of tissue morphology; and / or high signal-to-noise ratio and low error rate.

[0130] In certain aspects, STARmap can be used to study molecularly defined cell types and activity-regulated gene expression in mouse visual cortex, and can be expanded to larger 3D tissue blocks, enabling volumetric visualization of previously unavailable short-range and long-range spatial organization of cortical neurons. In some embodiments, the methods disclosed herein can be used to image DNA-conjugated antibodies for highly multiplexed protein detection.

[0131] The devices, methods and systems of the present invention can also be generalized to study many heterogeneous cell populations in various tissues. Without being bound by any scientific theory, STARmap analysis of the brain faces special challenges. For example, the expression of polymorphic activity-regulated genes (ARGs) observed in different cell types may depend on both the intrinsic biological properties of the cells (e.g., expression of signal transduction pathway components) and non-intrinsic properties (e.g., the anatomical structure of the neural circuits that transmit external sensory information to different cells (here refers to cells in the visual cortex)). In these cases, in situ transcriptome analysis, such as STARmap, can effectively link imaging-based molecular information with anatomical and activity information to clarify brain function and dysfunction.

[0132] The devices, methods and systems disclosed herein are capable of removing cellular components (e.g., lipids that typically provide structural support but hinder visualization of subcellular proteins and molecules) while maintaining the three-dimensional structure of cells and tissues because the sample is cross-linked with a hydrogel that physically supports the ultrastructure of the tissue. This removal renders the interior of the biological specimen substantially permeable to light and / or macromolecules, allowing for microscopic observation of the interior of the specimen (e.g., cells and subcellular structures) without the need for time-consuming, destructive tissue sectioning. This process is faster than the processes typically used in the art because the transparentization and permeabilization, which are typically performed in separate steps, can be performed together in a single step to remove cellular components. In addition, the specimen can be repeatedly stained, unstained, or re-stained with other reagents for comprehensive analysis. Further functionalization with a polymerizable acrylamide moiety allows for covalent anchoring of the amplicons at multiple sites within the polyacrylamide network.

[0133] In one example, the subject device, method, and system can be used to evaluate, diagnose, or monitor a disease. "Diagnosis" as used herein generally includes predicting a subject's susceptibility to a disease or condition; determining whether a subject is currently affected by a disease or condition; determining the prognosis of a subject affected by a disease or condition (e.g., determining the cancerous condition, cancer stage, and the likelihood that the patient will die from the cancer); predicting a subject's response to a treatment for a disease or condition (e.g., showing a positive response, a negative response, or no response at all to treatments such as allogeneic hematopoietic stem cell transplantation, chemotherapy, radiotherapy, antibody therapy, or small molecule combination therapy); and therapeutic determination (e.g., monitoring a subject's physical condition to provide information about the effect or efficacy of a treatment). For example, a biopsy section can be prepared from cancerous tissue and analyzed microscopically to determine the type of cancer, the extent of cancer progression, and whether the cancer responds to therapeutic intervention.

[0134] The apparatus, method and system of the present invention also provide a practical technique for screening candidate therapeutic agents to understand their effectiveness on tissues or diseases. For example, a subject (e.g., mouse, rat, dog, primate, human, etc.) can be exposed to a candidate agent, and then an organ or a biopsy thereof is prepared by a standard method, and the prepared sample is analyzed microscopically for one or more cell or tissue parameters. Parameters are quantifiable components of cells or tissues, especially components that can be accurately measured in high-throughput systems. Parameters can be any cell component or cell product, including cell surface determinants, receptors, proteins or their conformations or post-translational modifications, lipids, carbohydrates, organic or inorganic molecules, nucleic acids (e.g., mRNA, DNA, etc.) or parts or combinations thereof derived from such cell components. Although most parameters provide quantitative readings, in some cases, semi-quantitative or qualitative results are acceptable. Readings can include a single determined value, or can include an average, median, or variance, etc. Characteristically, a series of parameter readings can be obtained for each parameter derived from multiple identical determinations. The corresponding values ​​are expected to vary, and standard statistical methods and common statistical methods for providing single values ​​are used to obtain the value range of each parameter in the test parameter group. Thus, for example, one such method may include detecting cell activity, tissue vascularization, the presence or absence of immune cell infiltration, the effectiveness of changing disease progression, etc. In some embodiments, the screening includes comparing the analyzed parameters with parameters from a control or reference sample (e.g., a sample prepared in a similar manner from a subject not exposed to the candidate agent). The target candidate agents for screening include known and unknown compounds covering a variety of chemical classifications, mainly organic molecules, which may include organometallic molecules, inorganic molecules, gene sequences, etc. The target candidate agents for screening also include nucleic acids, for example, nucleic acids encoding siRNA, shRNA, antisense molecules or miRNA, or nucleic acids encoding polypeptides. An important aspect of the present invention is the evaluation of candidate drugs, including toxicity tests, etc. The evaluation of tissue samples using the target method may include, for example, gene, transcriptome, genome, proteome and / or metabolome analysis.

[0135] The devices, methods and systems of the present invention can also be used to visualize the distribution of genetically encoded markers throughout tissues at subcellular resolution, for example, chromosomal aberrations (inversions, duplications, translocations, etc.), loss of heterozygosity of genes, the presence of alleles indicating a predisposition to disease or good health, possible response to treatment, ancestry, etc. Such detection can be used, for example, for disease diagnosis and monitoring, personalized medicine, and parentage studies, such as those described above.

[0136] Can compile analytical information database.These databases can include the result from known cell type, the reference information from the analysis of the cell processed under specific conditions etc.Can generate data matrix, each point in wherein said data matrix corresponds to a reading derived from a cell, wherein the data of each cell can include the reading derived from a plurality of marks.Reading can be the mean value, median, variance or other statistical or mathematically derived value related to measurement.Reading by directly comparing corresponding reference, can further refine output reading information.Under the same conditions, the absolute value obtained for each output will show the variability inherent to living organisms, can also reflect the variability inherent between individual cell variability and individual. Examples of non-limiting aspects of the invention

[0137] The subject matter of the present invention described above (including embodiments) may be used alone or in combination with one or more contents or embodiments. Without limiting the foregoing, certain non-limiting contents of items 1-89 of this patent are provided below. After reading the present invention, the following contents will be apparent to those skilled in the art, and each individually numbered content may be used alone or in combination with any of the preceding or succeeding individually numbered contents. This is intended to provide support for all such combinations of the contents, and is not limited to the combinations of the contents explicitly provided below: 1. A method for in situ gene sequencing of a target nucleic acid in a cell in an intact tissue, the method comprising: (a) contacting the fixed permeabilized intact tissue with at least one pair of oligonucleotide primers under conditions that allow specific hybridization to occur, wherein the primer pair comprises a first oligonucleotide and a second oligonucleotide; wherein the first oligonucleotide and the second oligonucleotide each comprise a first region of complementarity, a second region of complementarity, and a third region of complementarity; wherein the second oligonucleotide further comprises a barcode sequence; wherein a first region of complementarity of the first oligonucleotide is complementary to a first portion of the target nucleic acid, wherein a second region of complementarity of the first oligonucleotide is complementary to a first region of complementarity of the second oligonucleotide, wherein a third region of complementarity of the first oligonucleotide is complementary to a third region of complementarity of the second oligonucleotide, wherein the second region of complementarity of the second oligonucleotide is complementary to a second portion of the target nucleic acid, and wherein the first region of complementarity of the first oligonucleotide is adjacent to the second region of complementarity of the second oligonucleotide; (b) adding a ligase to connect the second oligonucleotide and generate a closed nucleic acid circle; (c) performing rolling circle amplification in the presence of a nucleic acid molecule, wherein the operation comprises using the second oligonucleotide as a template and using the first oligonucleotide as a primer that causes a polymerase to form one or more amplicons; (d) encapsulating the one or more amplicons in the presence of a hydrogel subunit to form one or more hydrogel-encapsulated amplicons; (e) contacting the one or more hydrogel-embedded amplicons having a barcode sequence with a pair of primers under conditions that allow ligation, wherein the primer pair comprises a third oligonucleotide and a fourth oligonucleotide, wherein ligation is achieved only when the third oligonucleotide and the fourth oligonucleotide ligate to the same amplicon; (f) repeating step (e); and (g) imaging the one or more hydrogel-embedded amplicons to determine in situ genetic sequencing of target nucleic acids in cells within intact tissue. 2. The method according to aspect 1, wherein the primer pair is heat denatured before contacting the sample. 3. The method according to aspect 1 or 2, wherein the cells are present in a cell population. 4. The method according to aspect 3, wherein the cell population comprises a plurality of cell types. 5. The method of any one of aspects 1-4, wherein contacting the fixed permeabilized intact tissue comprises hybridizing the primer pair to the same target nucleic acid. 6. The method according to any one of aspects 1-5, wherein the target nucleic acid is RNA. 7. The method according to aspect 6, wherein the RNA is mRNA. 8. The method according to any one of aspects 1-5, wherein the target nucleic acid is DNA. 9. The method according to any one of aspects 1-8, wherein the second oligonucleotide comprises a padlock probe. 10. The method according to any one of aspects 1 to 9, wherein the first region of complementarity of the first oligonucleotide is 19-25 nucleotides in length. 11. A method according to any one of aspects 1-10, wherein the second region of complementarity of the first oligonucleotide is 6 nucleotides in length. 12. The method according to any one of aspects 1-11, wherein the third region of complementarity of the first oligonucleotide is 6 nucleotides in length. 13. A method according to any one of aspects 1-12, wherein the first region of complementarity of the second oligonucleotide is 6 nucleotides in length. 14. The method according to any one of aspects 1 to 13, wherein the second region of complementarity of the second oligonucleotide is 19-25 nucleotides in length. 15. A method according to any one of aspects 1-14, wherein the third region of complementarity of the second oligonucleotide is 6 nucleotides in length. 16. The method according to any one of aspects 1 to 15, wherein the first region of complementarity of the second oligonucleotide comprises the 5' end of the second oligonucleotide. 17. The method according to any one of aspects 1 to 16, wherein the third region of complementarity of the second oligonucleotide comprises the 3' end of the second oligonucleotide. 18. The method according to any one of aspects 1 to 17, wherein the first region of complementarity of the second oligonucleotide is adjacent to the third region of complementarity of the second oligonucleotide. 19. The method according to any one of aspects 1-18, wherein the barcode sequence of the second oligonucleotide provides barcode information for identifying the target nucleic acid. 20. The method of any one of aspects 1-19, wherein contacting the fixed permeabilized intact tissue comprises hybridizing a plurality of oligonucleotide primers specific for different target nucleic acids. 21. The method according to aspect 1, wherein the second oligonucleotide is provided in the form of a closed nucleic acid circle and the step of adding a ligase is omitted. 22. The method according to any one of aspects 1-21, wherein the melting temperature (Tm) of the oligonucleotide is selected to minimize ligation of the oligonucleotide in solution. 23. The method according to any one of aspects 1-22, wherein adding a ligase comprises adding a DNA ligase. 24. The method according to any one of aspects 1-23, wherein the nucleic acid molecule comprises amine-modified nucleotides. 25. The method of aspect 24, wherein the amine-modified nucleotide comprises an acrylic acid N-hydroxysuccinimide moiety modification. 26. The method of any one of aspects 1-25, wherein the embedding comprises copolymerizing the one or more amplicons with acrylamide. 27. The method of any one of aspects 1-26, wherein the embedding comprises rendering the one or more hydrogel-embedded amplicons transparent, wherein the target nucleic acid is substantially retained in the one or more hydrogel-embedded amplicons. 28. The method of aspect 27, wherein the clearing comprises substantially removing a plurality of cellular components from the one or more hydrogel-embedded amplicons. 29. The method of aspect 27 or 28, wherein the clearing comprises substantially removing lipids from the one or more hydrogel-embedded amplicons. 30. The method according to any one of aspects 1-29, wherein the third oligonucleotide is configured to decode a base. 31. The method according to any one of aspects 1-30, wherein the fourth oligonucleotide is configured to convert the decoded base into a signal. 32. The method of aspect 31, wherein the signal is a fluorescent signal. 33. The method of any one of aspects 1-32, wherein contacting the one or more hydrogel-embedded amplicons comprises eliminating accumulation of errors during sequencing. 34. A method according to any one of aspects 1-33, wherein the imaging comprises imaging the one or more hydrogel-embedded amplicons using confocal microscopy, two-photon microscopy, light-field microscopy, whole tissue expansion microscopy and / or CLARITYTM optimized monolayer light microscopy (COLM). 35. The method of any one of aspects 1-34, wherein the intact tissue is a thin section. 36. The method of aspect 35, wherein the intact tissue has a thickness of 5-20 μm. 37. The method of aspect 35 or 36, wherein contacting the one or more hydrogel-embedded amplicons occurs four or more times. 38. The method of aspect 35 or 36, wherein contacting the one or more hydrogel-embedded amplicons occurs five or more times. 39. The method of any one of aspects 1-34, wherein the intact tissue is a thick section. 40. The method of aspect 39, wherein the intact tissue has a thickness of 50-200 μm. 41. The method of aspect 39 or 40, wherein contacting the one or more hydrogel-embedded amplicons occurs six or more times. 42. The method of aspect 39 or 40, wherein contacting the one or more hydrogel-embedded amplicons occurs seven or more times. 43. A method for screening a candidate agent to determine whether the candidate agent can modulate gene expression of a nucleic acid in a cell within an intact tissue, the method comprising: (a) contacting the fixed permeabilized intact tissue with at least one pair of oligonucleotide primers under conditions that allow specific hybridization to occur, wherein the primer pair comprises a first oligonucleotide and a second oligonucleotide; wherein the first oligonucleotide and the second oligonucleotide each comprise a first region of complementarity, a second region of complementarity, and a third region of complementarity; wherein the second oligonucleotide further comprises a barcode sequence; wherein a first region of complementarity of the first oligonucleotide is complementary to a first portion of the target nucleic acid, wherein a second region of complementarity of the first oligonucleotide is complementary to a first region of complementarity of the second oligonucleotide, wherein a third region of complementarity of the first oligonucleotide is complementary to a third region of complementarity of the second oligonucleotide, wherein the second region of complementarity of the second oligonucleotide is complementary to a second portion of the target nucleic acid, and wherein the first region of complementarity of the first oligonucleotide is adjacent to the second region of complementarity of the second oligonucleotide; (b) adding a ligase to connect the second oligonucleotide and generate a closed nucleic acid circle; (c) performing rolling circle amplification in the presence of a nucleic acid molecule, wherein the operation comprises using the second oligonucleotide as a template and using the first oligonucleotide as a primer that causes a polymerase to form one or more amplicons; (d) encapsulating the one or more amplicons in the presence of a hydrogel subunit to form one or more hydrogel-encapsulated amplicons; (e) contacting the one or more hydrogel-embedded amplicons having a barcode sequence with a pair of primers under conditions that allow ligation, wherein the primer pair comprises a third oligonucleotide and a fourth oligonucleotide, wherein ligation is achieved only when the third oligonucleotide and the fourth oligonucleotide ligate to the same amplicon; (f) repeating step (e); (g) imaging the one or more hydrogel-embedded amplicons to determine in situ genetic sequencing of target nucleic acids in cells within intact tissues; and (h) detecting the gene expression level of the target nucleic acid, wherein a change in the expression level of the target nucleic acid in the presence of the at least one candidate agent relative to the expression level of the target nucleic acid in the absence of the at least one candidate agent indicates that the at least one candidate agent regulates gene expression of nucleic acids in cells within intact tissue. 44. A method according to aspect 43, wherein the primer pair is heat denatured prior to contacting the sample. 45. The method of aspect 43 or 44, wherein the cells are present in a cell population. 46. ​​The method of aspect 45, wherein the cell population comprises a plurality of cell types. 47. The method of any one of aspects 43-46, wherein contacting the fixed permeabilized intact tissue comprises hybridizing the primer pair to the same target nucleic acid. 48. The method of any one of aspects 43-47, wherein the target nucleic acid is RNA. 49. The method according to aspect 48, wherein the RNA is mRNA. 50. The method according to any one of aspects 43-47, wherein the target nucleic acid is DNA. 51. The method according to any one of aspects 43 to 50, wherein the second oligonucleotide comprises a padlock probe. 52. The method according to any one of aspects 43 to 51, wherein the first region of complementarity of the first oligonucleotide is 19 to 25 nucleotides in length. 53. A method according to any one of aspects 43 to 52, wherein the second region of complementarity of the first oligonucleotide is 6 nucleotides in length. 54. A method according to any one of aspects 43 to 53, wherein the third region of complementarity of the first oligonucleotide is 6 nucleotides in length. 55. A method according to any one of aspects 43-54, wherein the first region of complementarity of the second oligonucleotide is 6 nucleotides in length. 56. A method according to any one of aspects 43 to 55, wherein the second region of complementarity of the second oligonucleotide is 19 to 25 nucleotides in length. 57. A method according to any one of aspects 43 to 56, wherein the third region of complementarity of the second oligonucleotide is 6 nucleotides in length. 58. The method according to any one of aspects 43 to 57, wherein the first region of complementarity of the second oligonucleotide comprises the 5' end of the second oligonucleotide. 59. The method according to any one of aspects 43 to 58, wherein the third region of complementarity of the second oligonucleotide comprises the 3' end of the second oligonucleotide. 60. The method according to any one of aspects 43 to 59, wherein the first region of complementarity of the second oligonucleotide is adjacent to the third region of complementarity of the second oligonucleotide. 61. The method according to any one of aspects 43-60, wherein the barcode sequence of the second oligonucleotide provides barcode information for identifying the target nucleic acid. 62. The method of any one of aspects 43-61, wherein contacting the fixed permeabilized intact tissue comprises hybridizing a plurality of oligonucleotide primers specific for different target nucleic acids. 63. The method according to aspect 43, wherein the second oligonucleotide is provided in the form of a closed nucleic acid circle and the step of adding a ligase is omitted. 64. The method according to any one of aspects 43-63, wherein the melting temperature (Tm) of the oligonucleotide is selected to minimize ligation of the oligonucleotide in solution. 65. The method of any one of aspects 43-64, wherein adding a ligase comprises adding a DNA ligase. 66. The method of any one of aspects 43-65, wherein the nucleic acid molecule comprises amine-modified nucleotides. 67. The method of aspect 66, wherein the amine-modified nucleotide comprises an acrylic acid N-hydroxysuccinimide moiety modification. 68. The method of any one of aspects 43-67, wherein the embedding comprises copolymerizing the one or more amplicons with acrylamide. 69. The method of any one of aspects 43-68, wherein the embedding comprises rendering the one or more hydrogel-embedded amplicons transparent, wherein the target nucleic acid is substantially retained in the one or more hydrogel-embedded amplicons. 70. The method of aspect 69, wherein the clearing comprises substantially removing a plurality of cellular components from the one or more hydrogel-embedded amplicons. 71. The method of aspect 69 or 70, wherein the clearing comprises substantially removing lipids from the one or more hydrogel-embedded amplicons. 72. A method according to any one of aspects 43-71, wherein the third oligonucleotide is configured to decode a base. 73. A method according to any one of aspects 43-72, wherein the fourth oligonucleotide is configured to convert the decoded base into a signal. 74. The method of aspect 73, wherein the signal is a fluorescent signal. 75. The method of any one of aspects 43-74, wherein contacting the one or more hydrogel-embedded amplicons comprises eliminating accumulation of errors during sequencing. 76. A method according to any one of aspects 43-75, wherein the imaging comprises imaging the one or more hydrogel-embedded amplicons using confocal microscopy, two-photon microscopy, light-field microscopy, whole tissue expansion microscopy and / or CLARITYTM optimized monolayer light microscopy (COLM). 77. The method of any one of aspects 43-76, wherein the intact tissue is a thin section. 78. The method of aspect 77, wherein the intact tissue has a thickness of 5-20 μm. 79. The method of aspect 77 or 78, wherein contacting the one or more hydrogel-embedded amplicons occurs four or more times. 80. The method of aspect 77 or 78, wherein contacting the one or more hydrogel-embedded amplicons occurs five or more times. 81. The method of any one of aspects 43-76, wherein the intact tissue is a thick section. 82. The method of aspect 81, wherein the intact tissue has a thickness of 50-200 μm. 83. The method of aspect 81 or 82, wherein contacting the one or more hydrogel-embedded amplicons occurs six or more times. 84. The method of aspect 81 or 82, wherein contacting the one or more hydrogel-embedded amplicons occurs seven or more times. 85. A method according to any one of aspects 43-84, wherein the detection comprises performing flow cytometry; sequencing; probe binding and electrochemical detection; pH change; enzyme-induced catalysis bound to a DNA tag; quantum entanglement; Raman spectroscopy; terahertz technology; and / or scanning electron microscopy. 86. According to aspect 85, the flow cytometry is mass cytometry or fluorescence activated flow cytometry. 87. The method according to any one of aspects 43-86, wherein the detecting comprises performing microscopy, scanning mass spectrometry or other imaging techniques. 88. The method of any one of aspects 43-87, wherein the detecting comprises determining a signal. 89. The method of aspect 88, wherein the signal is a fluorescent signal. 90. A system comprising: a fluidic device comprising an imaging chamber and a pump; and A processor unit configured to implement any of aspects 1-42. Example

[0138] The following examples are presented so as to fully disclose and describe methods of making and using the invention to one of ordinary skill in the art and are not intended to limit the scope of what the inventors regard as their invention nor to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp: base pair; kb: kilobase; pl: picoliter; s or sec: second; min: minute; h or hr: hour; aa: amino acid; kb: kilobase; bp: base pair; nt: nucleotide; im: intramuscular; ip: intraperitoneal; sc: subcutaneous injection; etc. Materials and methods

[0139] Unless otherwise noted, the following materials and methods are generally applicable to the results in the examples described in this patent. Mouse

[0140] All animal procedures followed the animal care guidelines approved by the Administrative Panel on Laboratory Animal Care (APLAC) at Stanford University and the guidelines published by the National Institutes of Health. C57 / BL6 male mice (6-10 weeks old) were used for the experiments. For the dark / lighting treatment experiments, mice were first raised on a standard light cycle and then placed in a dark and lightless environment for 5 days. After the end of the dark environment, the mice were killed or exposed to light for 1 hour before being killed. For the cocaine experiments, mice were injected with saline or 15 mg / kg cocaine 1 hour before being killed. For thin sections, animals were anesthetized with isoflurane and quickly decapitated. Brain tissue was removed, placed in OCT, frozen in liquid nitrogen, and then sliced ​​using a cryostat (Leica CM1900; see the thin tissue section below for details). For large volume samples, animals were anesthetized with Buprenex (100 mg / ml, ip) and perfused transcardially with cold PBS followed by 4% PFA (see the large volume sample section below for details). Thy1-YFP mice were B6.Cg-Tg(Thy1-YFP)HJrs / J. Parvalbumin transgenic mice were generated by crossing Parv-IRES-Cre (JAX No.: 8069) and Ai14 (JAX No.: 7908) mice. Chemicals and enzymes

[0141] Chemicals and enzymes are listed by name (suppliers, catalog numbers): Gel Slick solution (Lonza, 50640). PlusOne affinity silane (GE Healthcare, 17-1330-01). Poly-L-lysine solution, 0.1% w / v (Sigma, P8920). Ultrapure distilled water (Invitrogen, 10977-015). Glass bottom 12-well and 24-well plates (MatTek, P12G-1.5-14-F and P24G-1.5-13-F). No. 2 micro coverslips, 12 mm diameter (Electron Microscope Sciences, 72226-01). OCT compound (Fisher, 23-730-571). 16% PFA, EM grade (Electron Microscope Sciences, 15710-S). Methanol for HPLC (Sigma-Aldrich, 34860-1L-R). PBS, 7.4 (Gibco, 10010-023 for 1×; 70011-044 for 10×). Tween-20, 10% solution (Calbiochem, 655206). Triton-X-100, 10% solution (Sigma-Aldrich, 93443). OminiPur formamide (Calbiochem, 75-12-7). 20× SSC buffer (Sigma-Aldrich, S6639). Vanadyl ribonucleoside complex (New England Biolabs, S1402S). Sheared salmon sperm DNA (Invitrogen, AM9680). SUPERase·In (Invitrogen, AM2696). T4 DNA ligase, 5 Weiss U / μL (Thermo Scientific, EL0011). Phi29 DNA polymerase (ThermoScientific, EP0094). 10 mM dNTP mix (Invitrogen, 100004893). BSA, molecular biology grade (New England Biolabs, B9000S). 5-(3-aminoallyl)-dUTP (Invitrogen, AM8439). BSPEG9 (Thermo Scientific, 21582). NHS acrylate, 90% (Sigma-Aldrich, A8060). NHS methacrylate, 98% (Sigma-Aldrich, 730300). Anhydrous DMSO (Molecular Probes, D12345). Acrylamide solution, 40% (Bio-Rad, 161-0140).Bis solution, 2% (Bio-Rad, 161-0142). Ammonium persulfate (Sigma-Aldrich, A3678). N,N,N',N'-tetramethylethylenediamine (Sigma-Aldrich, T9281). OminiPur SDS, 20% (Calbiochem, 7991). Proteinase K, RNA grade (Invitrogen, 25530049). Shrimp alkaline phosphatase (New England Biolabs, M0371L). DAPI (Molecular Probes, D1306). NeuroTrace fluorescent Nissl staining solution, yellow (Molecular Probes, N-21480). PMSF (Sigma, 93482). Papain (Worthington, LS003127). Artificial basement membrane (Corning Life Sciences, 356234). Neurobasal-A medium (Invitrogen, 21103-049). FBS (HyClone, SH3007103). B-27 supplement (Gibco, 17504044). 2mM Glutamax (Gibco, 35050-061). Fluorodeoxyuridine (Sigma, F-0503). Anti-NeuN antibody (Abcam, 190565). Primary mouse cortical neuron culture

[0142] On postnatal day 0 (P0) of mouse pups, the neocortex or hippocampus was removed from these pups, digested with 0.4 mg / mL papain, and then seeded at a density of 65,000 cells per well on a 24-well glass-bottom culture plate pre-coated with 1:30 artificial basement membrane. The cultured neurons were placed in Neurobasal-A medium containing 1.25% FBS, 4% B-27 supplement, 2mM Glutamax and 2mg / ml fluorodeoxyuridine, and then placed in a humidified incubator at 37°C and 5% CO2. smFISH

[0143] Stellaris ShipReady smFISH probe for mouse Gapdh with Quasar 570 was purchased from LGC Bioresearch Technologies (SMF-3002-1). smFISH experiments were performed according to the manufacturer's protocol for adherent cells and frozen mouse brain tissue. STARmap probe design

[0144] The SNAIL probes were designed as follows: (1) The common cDNA sequences (CCDS) of all mouse proteins were downloaded from ftp: / / ftp.ncbi.nlm.nih.gov / pub / CCDS / current_mouse, and the reference transcriptome information was downloaded (hgdownload.cse.ucsc.edu / goldenPath / mm10 / bigZips / ). For genes with multiple transcript isoforms, only the shortest isoform was considered; (2) Picky 2.2 (32 bits) was used to design the hybridization sequence of each probe pair, with a length limit of 40-46 nucleotides (nt); 4 sequences were designed for each gene; (3) The resulting complementary DNA (cDNA) sequence (40-46nt) was divided into two halves, each occupying 20-25nt, with a spacing of 0-2nt between the two, and the melting temperature (Tm) between the two was optimally matched. All probes were shorter than the length of the 60nt composition and were made into 96-well plates by Integrated DNA Technologies (IDT). For the 160-transcript experiment, the homemade sequencing reagents included six readout probes (R1 to R6) and sixteen 2-base coded fluorescent probes (2base_F1 to 2base_F16) labeled with Alexa 488, 546, 594, and 647. For the 28-transcript large-volume experiment, all SNAIL primer probes were ordered based on acrylamide modification and then sequenced with 11-nt orthogonal readout probes (OR1 to OR7) and four 1-base fluorescent probes (1base_F1 to 1base_F4). All sequences are provided as Excel data files on the publisher's website. STARmap program for cell cultures and thin tissue sections Sample preparation

[0145] Treat glass-bottom 12-well or 24-well plates with methacryloxypropyltrimethoxysilane (Bind-Silane). For brain tissue sections, 12-well plates were further treated with poly-L-lysine solution. Pre-treat No. 2 microcoverslips (12 mm) with GelSlick for subsequent polymerization according to the manufacturer's instructions. Primary neuronal cell cultures were fixed with 1.6% PFA in PBS for 10 minutes and then transferred to pre-cooled (-20°C) methanol and then placed at -80°C for at least 15 minutes (up to 1 week). For brain tissue, freshly excised mouse brains were immediately embedded in OCT and snap frozen. Tissues were stored at -80°C or transferred to a cryostat and cut into 16-μm sections. Sections containing primary visual cortex were placed on pre-treated glass-bottom culture plates. Brain sections were fixed with 4% PFA in PBS for 10 min at room temperature, permeabilized with -20°C methanol, and then placed at -80°C for 15 min before hybridization. Library construction

[0146] The SNAIL probe was dissolved in RNase-free ultrapure water to a concentration of 100 or 200 μM and then mixed. The probe mixture was heated at 90°C for 2 to 5 minutes and then allowed to cool at room temperature. The sample was taken at -80°C, equilibrated to room temperature for 5 minutes, washed with PBSTR (0.1% Tween-20, 0.1U / μL SUPERase·In dissolved in PBS) for 2-5 minutes, and then placed in a 40°C humidified oven containing 1X hybridization buffer (2X SSC, 10% formamide, 1% Tween-20, 20mMRVC, 0.1mg / ml salmon sperm DNA and mixed SNAIL probe (100nM for each oligonucleotide)) and incubated with gentle shaking overnight. Then, the sample was washed twice (20 minutes) with PBSTR at 37°C, followed by washing once (20 minutes) in 4X SSC, and then dissolved in PBSTR. Finally, the sample was briefly rinsed once with PBSTR at room temperature. Then, the sample was incubated and gently stirred for 2 hours at room temperature with T4 DNA ligation mixture (1:50 dilution of T4 DNA ligase diluent, with 1X BSA and 0.2U / μL SUPERase-In added). Then, the sample was washed twice with PBSTR, followed by incubation and stirring for 2 hours at 30°C with RCA mixture (1:50 dilution of Phi29 DNA polymerase diluent, 250μM dNTP, 1XBSA, 0.2U / μL SUPERase-In, and 20μM 5-(3-aminoallyl)-dUTP). Next, at room temperature, the sample was washed twice in PBST (PBSTR, without SUPERase·In), and then treated with a solution of 20mM acrylic acid NHS ester dissolved in PBST for 2 hours. At room temperature, the sample was briefly washed once with PBST and then incubated with monomer buffer (4% acrylamide, 0.2% bisacrylamide, 2X SSC) for 30 minutes. The buffer was aspirated, and then 10 μL of polymerization mixture (0.2% ammonium persulfate, 0.2% tetramethylethylenediamine dissolved in monomer buffer) was added to the center of the sample, and then immediately covered with a cover glass coated with Gel Slick, incubated at room temperature for 1 hour, and then washed twice with PBST for 5 minutes each. The tissue gel hybrid was digested with proteinase K (0.2 mg / ml) at 37°C for 1 hour, and then washed 3 times with PBST (5 minutes each). Imaging and sequencing

[0147] For single gene detection, 19-nt fluorescent oligonucleotides complementary to the DNA amplicon were diluted in 1X SSC in PBST to a concentration of 500 nM, and the samples were then incubated at room temperature for 30 minutes and then washed three times with PBST for 5 minutes each before imaging. For sequencing, each sequencing cycle began with treating the samples twice (10 minutes) with stripping buffer (60% formamide, 0.1% Triton-X-100) at room temperature, followed by three washes with PBST for 5 minutes each. The samples were incubated for 3 hours at room temperature with sequencing mixture (1X T4 DNA ligase buffer, 1:25 dilution of T4 DNA ligase diluent, 1X BSA, 10 μM reading probe, and 5 μM fluorescent oligonucleotide). Then, before imaging, the samples were rinsed three times (10 minutes) with washing and imaging buffer (2X SSC and 10% formamide). DAPI staining was performed according to the manufacturer's instructions before the first cycle and after the sixth cycle to align the sequencing images with Nissl staining. After the sixth cycle, Nissl staining was performed for cell segmentation according to the manufacturer's instructions. Images were acquired using a Leica TCS SP8 confocal microscope equipped with a 405 diode, white light laser, 40x oil immersion objective (NA 1.3), with a voxel size of 78 nm × 78 nm × 315 nm. Thin-section STARmap data processing

[0148] All image processing steps were implemented using MATLAB R2017A. Image Registration

[0149] Image registration is done using a 3D Fast Fourier Transform (FFT) to compute the cross-correlation between the two image volumes at all translation offsets. The location of the maximum correlation coefficient is determined and used to translate the image volume to compensate for the offset. All images are registered based on the first round of sequencing, first globally for the entire field of view and then for each tile (corresponding to each tile field of view used by the microscope during image acquisition). Point recognition

[0150] After registration, individual spots were identified in each color channel based on the first round of sequencing. For the 160-gene experiment, spots with a diameter of approximately 7 pixels were identified by first filtering the image volume with a 3D Laplacian of Gaussian filter and then determining local maxima in 3D. For the 1020-gene experiment, spots were identified in a similar way, but the Laplacian of Gaussian was calculated at multiple scales and the maxima were also determined at these scales. After each spot was identified, in each round, the dominant color of the spot in all four channels was determined for a 3x3x1 voxel volume surrounding the spot location. Barcode filtering

[0151] Spots are first filtered based on a quality score. The quality score quantifies the probability that each spot is from one color rather than a mixture of colors in each sequencing cycle. The barcode code blocks are converted to color space based on the expected color sequence obtained after 2-base encoding of the barcode DNA sequence. Spot color sequences that meet the quality threshold and match the sequence in the code block are retained and identified with the specific gene represented by the barcode; all other spots are rejected. High-quality spots and associated gene identities in the code block are then saved for downstream analysis. 2D cell segmentation

[0152] After the last round of sequencing, nuclei were manually identified based on the maximum intensity projection of the DAPI channel. Cell bodies were first identified based on Nissl staining using the Random Forests classifier used in Ilastik. The classifier was trained with a subset of cropped regions randomly selected from all samples and then applied to the entire image. The thresholded probability map was then pixel-dilated to fill the remaining holes. Finally, based on the merged thresholded cell body map and the determined nucleus position, a marker-based watershed transform was used to segment the thresholded cell bodies. Convex hull calculations were performed around each cell body. Points that overlapped with each convex hull in 2D were then assigned to the cell to calculate the gene expression matrix for each cell. Single cell data preprocessing

[0153] All single-cell analyses were performed using a custom package in Python for STARmap experimental analysis. First, the expression matrix for each cell was normalized to the expression values ​​Eij of all genes j for each cell i using the following formula:

[0154] N ij =ln(1+median(E i: )*(E ij / ΣE i: ))

[0155] For clustering, effects related to the number of transcripts per cell, sample identity, and experimental conditions (light vs. dark treatment) were regressed using a linear model:

[0156] E ij =nSpots i +exptID i +exptCond i , and assume that E ij It conforms to Poisson distribution. Top single cell clustering

[0157] After normalization and scaling, principal component (PC) analysis was performed to reduce the dimensionality of the cell expression matrix. The top PCs were used for top clusters based on a manual analysis of the explained variance ratios of each PC based on the explained variance ratios. The top PCs were then clustered using the shared nearest neighbor (SNN) algorithm and Louvain distance. Clusters enriched for the excitatory neuron marker Slc17a7 (vesicular glutamate transporter), inhibitory neuron marker Gad1, and non-neuronal marker Mqp were manually merged to form three major clusters representing these cell types. Cells were visualized using unified manifold approximation and projection (UMAP) (https: / / github.com / lmcinnes / umap). Cells in each cluster were then sub-clustered using PCA decomposition, followed by Louvain SNN clustering to identify specific cell types. Single cell subclustering

[0158] The inhibitory, excitatory, and non-neural clusters were sub-clustered using the same approach as applied to the main clusters. Differential expression analysis

[0159] The bimod test (likelihood ratio test) was used to calculate the P value of differential expression of each gene between each cluster and all other clusters, thereby selecting genes that are specifically variable in the sub-cluster. The P value was FDR corrected according to the number of clusters. STARmap program for thick tissue sections Sample preparation

[0160] Glass-bottom 12-well plates and micro coverslips (12 mm) were pretreated in the same manner as thin tissue sections. Mice were perfused with PFA and then postfixed on ice for 2-3 hours, then transferred to PBS on ice for 30 minutes and cut into 150-μm sections. Sections containing primary visual cortex were transferred to pretreated glass-bottom culture plates and washed once with ice-cold PBS. Library construction

[0161] Samples were pre-cleared with -20°C chilled methanol for 1 hour at 4°C and then incubated in a 40°C humidified oven with 1× permeabilization and hybridization buffer (2X SSC, 10% formamide, 1% Triton-X-100, 20mM RVC, 0.1mg / ml salmon sperm DNA, mixed SNAIL probes (100nM each oligonucleotide), and 0.2% SDS) with gentle shaking for two days. Methanol treatment prevents sample swelling and deformation; for co-detection of protein and RNA (Thy1-YFP), methanol pre-clearing was skipped. Samples were washed twice with PBSTV (0.1% Triton and 2mM RVC) for 1 hour at 37°C, and then washed once with PBS for 1 hour. Next, samples were incubated with polymerization mixture I (4% acrylamide, 0.2% bisacrylamide, 2X SSC, 0.5% VA-044) at 4°C for 1 hour. The buffer was aspirated, and then 40 μL of polymerization mixture II (0.1% ammonium persulfate, 0.1% tetramethylethylenediamine dissolved in polymerization mixture I) was added to the center of the sample, and then immediately covered with a cover glass coated with Gel Slick and incubated at 40°C for 1 hour. Then, the sample was washed twice with PBSTV for 20 minutes each time. The tissue gel hybrid was digested overnight at 37°C with proteinase K (0.2 mg / ml dissolved in 2XSSC, 1% SDS), and then washed twice with PBSF (2mM PMSF dissolved in PBS) and PBSTR (30 minutes each time). Next, the sample was incubated and gently stirred for 12 hours at room temperature with T4 DNA ligation mixture (1:50 diluted T4 DNA ligase diluent, and 1X BSA and 0.2U / μL SUPERase-In were added). Then, the samples were washed twice with PBSTR (1 hour each time) and then incubated with RCA mixture (1:50 dilution of Phi29 DNA polymerase, 250 μM dNTP, 1X BSA, 0.2 U / μL SUPERase-In and 20 μM 5-(3-aminoallyl)-dUTP) at 30° C. for 12 to 24 hours. Finally, the samples were cross-linked by BSPEG9. Imaging and sequencing

[0162] First, the samples were stained with DAPI overnight and then rinsed with PBS for 1 hour. Each cycle began with treating the samples twice (20 minutes) with stripping buffer (60% formamide, 0.1% Triton-X-100) at room temperature, followed by washing 3 times with PBST for 20 minutes each. The samples were incubated with a large amount of sequencing mixture (1X T4 DNA ligase buffer, 1:50 diluted T4 DNA ligase diluent, 1X BSA, 5μM orthogonal read probes, and 400nM 1 base fluorescent oligonucleotides) at room temperature for 4 hours. The samples were then rinsed twice with PBS (20 minutes each) and then imaged. Images were collected using a Leica TCS SP8 confocal microscope equipped with a 405nm diode, a white light laser, and a 25x water immersion objective (NA 0.95), with a voxel size of 0.9μm×0.9μm×1μm. Large-volume STARmap data processing

[0163] The images were again registered (3D FFT registration), except for the case where the DAPI channel was used for registration. Specifically, the DAPI channel in each round was globally registered based on the first round, and then segment-by-segment registered in a 4×5 grid corresponding to the field of view tile used to acquire the image. Cell identification and quantification

[0164] After registration, cells were identified using the minima of a Laplacian of Gaussian filter applied to the DAPI channel. To quantify the expression of each gene, the mean intensity of each color channel was averaged for a 10x10x3 voxel volume surrounding each cell nucleus. 3D Cell Analysis

[0165] First, by K-means clustering, Gad1, Slc17a7 and several non-neuronal genes were used to cluster cells into inhibitory, excitatory and non-neuronal clusters. Then, each cluster was sub-clustered by K-means clustering. The initial value of K-means clustering was set to the average expression value of each marker gene. In order to calculate the distance between cell types, a kD tree (suitable for fast nearest neighbor calculation using Euclidean distance) was used to calculate the nearest neighbor distance between cells in all excitatory neurons and each inhibitory neuron subtype. Assessing physical constraints of STARmap using the Actb insertion gene

[0166] The 100 and 1,000 genes had a common 18nt sequence in the padlock (sequence A) that would be amplified in the final nanospheres. Another set of SNAIL probes was designed for Actb with a different 18nt sequence (sequence B). The SNAIL probes for Actb were mixed with SNAIL probes for 0, 100, or 1,000 genes and used for hybridization. The Actb insert gene and the 100 and 1,000 genes all went through the same ligation and amplification steps to ensure efficiency consistency. For readout, two fluorescent detection oligonucleotides (an Alexa488 probe complementary to sequence B and an Alexa 647 probe targeting sequence A) were added to the sample; the amplicon for Actb and the amplicons for the remaining genes were imaged in two independent channels. Then, the amplicon for Actb RNA was tested to determine whether the number of amplicons could be diluted by increasing the number of other genes as an indication of molecular crowding ( Figures 16A-16E ). Immunostaining of clarified tissue

[0167] PFA-fixed tissues were processed as described previously in the corresponding publication of Shendure et al. (2005). Briefly, PFA-fixed brain sections with a thickness of 200 μm were placed in 1% acrylamide embedding solution at 4°C for 23 hours, then embedded at 37°C for 4 hours, and then passively cleared for 5 days. Cleared sections were washed in PBST for 2 days, then stained with anti-NeuN (1:100) for 24 hours at room temperature, and then washed in PBST for 24 hours. Sections were imaged using confocal microscopy. Automated in situ sequencing equipment (expected)

[0168] The automated steps of the experiment involve three buffers that are kept in bulk at room temperature: 1) Wash Buffer, 2) Imaging Buffer, and 3) Stripping Buffer. A tube containing the ligase mix will be kept at 4°C. During the experiment, there will be 8 tubes containing fluorescently labeled imaging materials, which are also kept at 4°C. Six of the tubes will contain fluorescently labeled oligonucleotides and probes for hybridization. One tube will contain the small molecule Nissl stain and another tube will contain the small molecule DAPI stain. If it is not possible to mix the ligase and oligonucleotides at the time of imaging, they can be pre-mixed and stored in an environment maintained at 4°C.

[0169] The sequencing process involves placing the sample in a custom imaging chamber and connecting to the fluidics system. In each of the six rounds of sequencing, the ligase mix and any of the six oligonucleotide mixes are mixed (total volume of 200-400 μl) and then treated with them for 3 hours. Next, wash buffer is flowed through the sample for 10 minutes. The wash buffer is then replaced with imaging buffer. The system then sends a trigger signal to the microscope, which performs fluorescent imaging. After imaging is complete, the microscope sends a trigger signal to the system to start the next round. The system flows stripping buffer through the sample for 10 minutes. Then, wash buffer is flowed for 5 minutes to remove residual stripping buffer. Ligase and fluorescently labeled oligonucleotides are used in the next round. After repeating this process six times, the laser wheel is stripped and washed, and then treated with Nissl and DAPI solutions for one hour each. The device is then washed and imaged in imaging buffer. Finally, after the experiment is completed, the entire system should be rinsed with bleach to clean it for the next use.

[0170] A computer controlled fluidics device can pump out the various solutions, mix the ligase with the oligonucleotide solution, and select the oligonucleotide solution to be used in each round. All parameters in this sequence (flow rate, time, etc.) should be programmable.

[0171] For whole tissue, a custom imaging chamber seals the tissue sections in a small volume with inflow and outflow ports and is manipulated using a microscope equipped with a 24-well plate holder. Fig.24A describes the overall hardware configuration used for STARmap sequencing, Fig. 24B Designs of exemplary fluid systems described herein are described. result Example 1: STARmap

[0172] The methods disclosed herein (including in situ sequencing techniques) can perform highly multiplexed gene detection (up to 1000 genes) in intact biological tissues. The methods start from primary biological tissues (fresh or preserved tissues, minimum size: one cell; maximum size: millimeters), which can output image-based gene quantification at subcellular and cellular resolution with high efficiency, low error rate and short processing time.

[0173] For a schematic diagram of the STARmap described in this paper, see Figure 1A After brain tissue preparation (see the Methods section for mouse brain acquisition protocol), custom SNAIL probes ( Figure 2A), which is enzymatically replicated as a cDNA amplicon. The amplicon is constructed in situ by partial modification with acrylic acid N-hydroxysuccinimide, and then copolymerized with acrylamide to be embedded in the hydrogel network (wavy line), followed by removal of unbound lipids and proteins ( Figures 3A-3F Each SNAIL probe contains a unique gene identifier fragment that is read out by in situ sequencing and 2-base coding for error correction (SEDAL, Figures 4A-4L ). Finally, highly multiplexed RNA quantification in 3D reveals gene expression and cell types in space.

[0174] Figure 1A The following method for SNAIL is described: a pair of primers and padlock probes amplifies the target-specific signal and removes the noise typically generated by nonspecific hybridization of a single probe. Figure 1C and 1D Indicates that only adjacent binding of primer and padlock probe can amplify the signal. mRNA A represents Gapdh and mRNA B represents Actb. Fluorescence images all show labeling of Gapdh mRNA (grey) and nuclei (blue) in mouse brain sections; note the absence of labeling due to primer and padlock mismatch ( Figure 1D , right). Scale bar is 10 μm. Figure 1E Describes in situ sequencing of DNA amplicons in tissue hydrogel complexes by SEDAL, a modified sequencing-by-ligation method designed for STARmap. For each cycle, the reading probe (lines without asterisks) contains increasing lengths of degenerate bases (N represents an equal mixture of A, T, C, and G) and phosphates at the 5' end (5'P) to set the reading position; the decoding probe (lines with asterisks) is labeled with a fluorophore and has a color code for the dinucleotide at the 3' end. Only when the two probes are fully complementary to the DNA template (lower sequence) are they ligated to form a stable product with a higher melting temperature, allowing subsequent imaging after washing away the unligated probes. After each imaging cycle, the probes are stripped from the tough tissue hydrogel using 60% formamide to start the next cycle. X indicates an unknown base to be read; "underline" indicates the decoding sequence; Ch1-4 indicates the fluorescence channel. Scale bar, 2 μm. Example 2: SNAIL

[0175] Reverse transcription can be the main efficiency-limiting step in in situ sequencing, and SNAIL can bypass this step using a designed pair of primers and padlock probes ( Figure 2A), which is only valid if both probes hybridize to the same RNA molecule. The design of SNAIL probes (a component of STARmap) involves the following: Design of each primer or padlock probe (19-25 nucleotides; nt, blue) m (nucleic acid melting temperature) is 60°C or higher to hybridize with the target RNA, while for T m Below room temperature for each test group, the complementary sequence between the primer and the padlock was only 6 nt, so at 40 °C, during DNA-RNA hybridization, primer-padlock DNA-DNA hybridization was negligible, but allowed DNA ligation with T4 DNA ligase in the next step.

[0176] The padlock probes are circularized and subjected to rolling circle amplification to generate cDNA nanospheres (amplicons) containing multiple copies of the cDNA ( Figures 1A-1D ). This mechanism ensures amplification of target-specific signals and removes the noise that is inevitably generated by nonspecific hybridization of a single probe. In fact, the absolute intensity and signal-to-noise ratio (SNR) under this mechanism are much higher than those of commercially available single-molecule fluorescence in situ hybridization (smFISH) probes ( Figure 2B-2F ). Figure 2B Depicted is a comparison of the signal-to-noise ratio (SNR; mean intensity of signal spots / mean intensity of background) of commercially available smFISH probes and SNAIL probes targeting Gapdh mRNA in mouse cortical cell cultures and mouse visual cortex slices. Error bars represent the standard deviation (sd) of spot intensity. Error bars represent the sd of 39,398 pixels, 30,297 pixels, 97,555 pixels, and 19,392 pixels corresponding to RNA signals derived from 640,000 pixels in the acquired images; ***P<0.0001, Student's t-test. Figure 2C-2F Cortical cell cultures are described ( Figure 2C and 2D ) and visual cortex slices ( Figure 2E and 2F ) Figure 2C and 2E ) and SNAIL probes ( Figure 2D and 2F ); scale bar is 10 μm. Figure 2GA comparison of multiplexed RNA imaging methods using rolling circle amplification (RCA) is described. SNAIL probes overcome the efficiency-limiting step of reverse transcription and greatly simplify the experimental workflow compared to FISSEQ and padlock probes; PLAYR requires four probes, an additional step, and two ligation sites, while SNAIL requires only one probe pair and one ligation site. Box plots of 151 cell-type gene markers measured in RNA per cell by single-cell RNA sequencing (scRNA-seq, ref) and STARmap (a 160-gene atlas extracted from the visual cortex) ( Figure 2G ). Box plots depict the first and third quartiles; center line indicates median; whiskers indicate 5% and 95% data points; P values ​​indicate rank sum test. For an overview of single-cell RNA sequencing and RCA-based multiplexed RNA detection methods, some figures were extracted from references ( Fig.2I ). Example 3: cDNA amplicons embedded in a tissue hydrogel environment

[0177] To embed cDNA amplicons into the tissue hydrogel environment, amine-modified nucleotides were incorporated into the rolling circle amplification reaction, which was then functionalized with acrylamide moieties (using N-hydroxysuccinimide acrylate) and copolymerized with acrylamide monomers to form the hydrogel ( Figure 1A , Figure 3A ). For a schematic diagram of the hydrogel histochemistry method used for STARmap in thin tissue sections, see Figure 3A DNA amplicons were synthesized in the presence of small amounts of 5-(3-aminoallyl)-dUTP, where T was altered to a lower rate and functionalized with polymerizable acrylamide moieties using acrylic acid N-hydroxysuccinimide ester (AA-NHS) to covalently anchor the DNA amplicons at multiple sites within the polyacrylamide network ( Figure 3A , right). The resulting tissue hydrogel was then subjected to protein digestion and lipid removal to improve transparency ( Figure 3B-3E ). Fluorescence image (total intensity from all four fluorescence channels) showing the results of 160-gene detection in mouse visual cortex ( Figure 3B and 3C ) and the results of 16 gene detection in the medial habenular nucleus of mice ( Figure 3D and 3E ). Compared with the untreated sample ( Figure 3B and 3D ), compared with the sample treated with 5-(3-aminoallyl)-dUTP HTC and depleted of lipids and proteins ( Figure 3C and 3E) with reduced opacity and autofluorescence. Scale bar 50 μm. DNA gel crosslinking allows DNA amplicons to be retained in the gel. A 160-gene sample was prepared with or without AA-NHS and imaged in the medial prefrontal cortex ( Figure 3F-3G Compared to the AA-NHS-treated sample, the freshly prepared sample without AA-NHS treatment showed a 36% signal loss, and after 24 h of storage at room temperature, a further 40% signal loss occurred, while the signal change of the AA-NHS-treated sample was only 9% ( Figure 3F ). Fluorescence intensity is the average of four technical replicates, and the imaging size is 120μm×120μm×3μm. Error bars are shown as mean ± standard deviation; ***p<0.001, two-sided t test. For description of fluorescence images, see Figure 3G , scale bar is 3.5 μm.

[0178] Because this designer chemistry requires the amplicons to be covalently linked to the hydrogel network, the position and shape of the target can be maintained through multiple detection cycles. Figure 3H Depicted is a magnified fluorescence image of a neuron in the visual cortex, which was detected by STARmap for Gapdh RNA (cycle 1) and stripped at cycle 2; images of cycles 1 and 2 are also merged to show the stable spatial position of the DNA amplicons during the sequencing cycles. Scale bar 2 μm. Example 4: SEDAL

[0179] A 5-base barcode (library size 1,024) was designed and embedded in each padlock probe as a unique gene identifier to be sequenced, thus enabling multiplexed gene detection ( Figure 1A ). The sequencing-by-synthesis paradigm was avoided because it requires higher reaction temperatures, which in turn affects imaging and sample stability compared to sequencing-by-ligation methods performed at room temperature. However, no reported or commercially available sequencing-by-ligation methods have been able to achieve the necessary SNR or accuracy for this challenging intact tissue application ( Figures 4A-4L Therefore, a new method called SEDAL was designed for STARmap ( Figures 4A-4L ).

[0180] For a schematic diagram of SEDAL, see Figure 4ASEDAL involves a T4 DNA ligase whose activity is severely hampered by base mismatches, and two sequencing probes: a reading probe that sets the base position to be interrogated, and a fluorescent decoding probe that converts the base information into a color for imaging. Unlike other ligation sequencing methods that use pre-annealed reading probes (or equivalent), the reading probes in SEDAL are shorter (11 nt, T m near room temperature), partially degenerate (e.g. Figure 4A As shown, for the 4th cycle, the first two bases at the 5' end are N, where N is an equal mixture of A, T, C, and G), and are mixed with a decoding probe and T4 DNA ligase to achieve a one-step reaction. At room temperature, the reading probe remains in a dynamic annealing state with the DNA template, and the probe is detached from the DNA template. Only when the reading probe perfectly matches the DNA template will the T4 DNA ligase connect to the fluorescent 8-nt decoding probe. In other words, only when a perfect match occurs will the two probes connect to form a stable product for imaging. The short reading code and decoding probe are then washed to allow the fluorescent 19-nt product to stably hybridize with the DNA amplicon for imaging. For the next cycle, the previous fluorescent product is stripped off, and the reading probe also includes a degenerate base that can move the reading frame by one base ( Figure 1E ). 5'P indicates 5' phosphate. 3'InvT indicates 3' inverted dT base, which can prevent the reading probe from self-ligating; 3'OH indicates 3' hydroxyl.

[0181] After each cycle corresponding to a base read, the fluorescent product is stripped with formamide, which eliminates the accumulation of errors as sequencing proceeds ( Figure 1E and Figure 4B ). For a comparison of key features of all sequencing-by-ligation methods, see Figures 4C-4F For background issues related to commercially available SOLiD sequencing kits when applied to mouse brain tissue, see Figure 4C and 4D , and the fewest background issues associated with custom SEDAL reagents are listed in Figure 4E and 4F . Signal image ( Figure 4C and 4E ) indicates the first sequencing cycle of Malat1, Actb, Calm1 and Snap25 genes. Background image ( Figure 4D and 4F ) was collected after the first cycle of cleavage / stripping operation was completed. The scale bar is 50 μm.

[0182] Schematic diagram of the 1-base and 2-base coding paradigms and example results with and without single sequencing errors (color errors) are shown in Figure 4G and 4HFor a 1-base code, a single sequencing error can result in a single base mutation, which in turn can result in a 5-base code being sequenced incorrectly ( Figure 4G ). For 2-base coding, the six-cycle paradigm plays a role in reducing errors. Since a single error in any sequencing cycle will be propagated and cause the adjacent known base G to mutate to other bases, the erroneous reads are rejected ( Figure 4H ). A 2-base encoding scheme was designed and implemented to reduce any residual errors associated with high-density spot imaging ( Figure 4G-4H ).

[0183] Based on a test set consisting of four highly expressed test genes, STARmap's error rate is more than an order of magnitude lower than that of existing methods (about 1.8% vs. 29.4%; Figure 4I-4L ). Actual data from cPAL (representing a 1-base coding scheme) and SEDAL (representing a 2-base coding scheme) were used for a 4-gene assay in the mouse visual cortex. The SNAIL probes for Malat1, Actb, Calm1, and Snap25 were identical in both cases, and the Hamming distance for each of the four 5-base codon pairs was 5 (i.e., complete non-homology); this sparse coding allowed an estimate of the sequencing error rate based on the fraction of erroneous spots (not containing the four 5-base codons used) among all detected spots. ( Figures 4I-4J ). For spatial plots of the four genes detected by cPAL and SEDAL, see Fig. 4I and Figure 4J . Error rate of cPAL in 4-gene experiment ( Fig. 4I ) is 29.4%( Figure 4K ), while the error rate of SEDAL ( Figure 4J ) in the built-in error reduction ( Figure 4L ) is 1.8%. Example 5: Cell classification of cell types in primary visual cortex using STARmap

[0184] To test whether STARmap could achieve its initial goal of high-content 3D whole-tissue sequencing of single-cell transcriptional states with the necessary sensitivity and accuracy, we applied STARmap to one of the most pressing challenges in neuroscience: detecting and classifying cell types and following the organizational architectural principles of the adult mouse neocortex. The anatomy and function of the mouse primary visual neocortex has been extensively studied, and results can be validated by comparison with previous findings across multiple papers, methodologies, and data sources, but the diversity of deeply molecularly defined cell types within the visual cortex has not been spatially resolved in a single experiment, making it impossible to identify underlying fundamental joint statistics and organizational principles across 3D volumes. In one of many examples of experimental leverage that such information can provide, the joint 3D cell type atlas was used to help decode the spatiotemporal logic of gene expression triggered by neural activity as a function of cell type and spatial location.

[0185] Primary visual cortex (V1) was sectioned coronally and six rounds of in situ SEDAL sequencing were performed on coronal mouse brain sections using a 5-base barcoded SNAIL probe ( Figure 1A , Figure 5A-5B ) to investigate a large curated gene set of 160 genes (including 112 putative cell type markers, curated from single-cell RNA sequencing of the mouse cortex) and 48 activity-regulated genes (ARGs). In one test cohort of mice, visually evoked neural activity was induced by 1 hour of light exposure after four days of dark housing; other mice were kept in continuous darkness before sacrifice. 8 μm-thick sections containing 600-800 cells were imaged involving all cortical layers. Figure 5B Depicts the raw fluorescence images of STARmap during the process, including a full view of cycle 1 ( Figure 5B , top) and a zoomed-in view of all six cycles ( Figure 5B , lower panel). Full view represents a view of 1.4 mm × 0.3 mm, and the scale bar is 100 μm; magnified area represents an area of ​​11.78 μm × 11.78 μm, and the scale bar is 2 μm; channels represent the color code applied to the four fluorescent channels; L1-6 represent the six neocortical layers; cc represents the corpus callosum; HPC represents the hippocampus.

[0186] After six rounds of sequencing, the cell bodies were segmented by fluorescent Nissl staining, so that the amplicons could be attributed to individual cells ( Figure 6A-6B ). Fig. 6AA simplified diagram of the processing pipeline for extracting decoded reads from raw imaging data is depicted (see also the Methods section for details on each step): (1) the sample is imaged over multiple rounds; (2) the sample is registered in each round, with two rounds (green and purple) experiencing misalignment that must be corrected by registration; (3) spots are automatically identified (independently in the first round) as putative amplicons in each color channel, which are decoded based on the color value of that spot in each round; (4) reads are identified by comparing the maximum intensity of each spot in each channel in each round to the predicted color sequence of each barcoded DNA sequence (barcode encoded in color space); (5) cells are detected using a machine learning-based segmentation approach that takes into account various intensity and texture features to segment Nissl-containing cells from the background (see ( for details). Figure 6B )); and (6) assigning the reads to cells by calculating the overlap between the position of each valid read and the convex hull of each cell segmentation region.

[0187] Genes encoded by reads overlapping the convex hull of a cell are assigned to that cell. Figure 6B A method for determining cell-related context is described: (1) a random forest classifier (a nonparametric machine learning algorithm for marker prediction) is trained on a downsampled set of Nissl staining data to distinguish regions containing cells from background; (2) cell locations are manually selected using the DAPI (nuclei) channel; (3) the classifier is applied to the entire image to predict cell locations; and (4) cells are segmented based on this prediction using a marker-based watershed that segments the cell-marked region of the image into discrete cell bodies based on the known locations of the nuclei.

[0188] Figure 5C The histogram in gives the reads (DNA amplicons) for each cell ( Figure 5C , left) and genes per cell ( Figure 5C , right). The values ​​corresponding to the amplicon for each cell and the gene for each cell have basically changed ( Figure 5C ), while the 160-gene expression patterns between biological replicates were consistent (R = 0.94-0.95, Figure 5D ), demonstrating that transcript diversity can be reliably detected at the single-cell level. For quantitative reproducibility of biological replicates, see Figure 5D , both under light and dark conditions: log2 (amplicon number) correlation images of 160 genes in the entire imaging area are plotted. Rep1 represents the expression value in the first replicate, and rep2 represents the expression value in the second replicate.

[0189] This 160-gene experiment faithfully reproduces the spatial distribution of known cortical layer markers and interneurons, illustrated here by comparing in situ images derived from paired public atlases with STARmap results ( Figure 5E ). Figure 5E The STARmap validation shown in describes the in situ images provided by the Allen Institute of Brain Science (AIBS) ( Figure 5E , left column) and RNA patterns of individual genes extracted from the 160-gene STARmap, which reliably reproduced the spatial gene expression patterns of AIBS ( Figure 5E , right column).

[0190] Cell classification was performed using expression data of 112 cell type markers. First, after principal component decomposition, more than 3,000 cells from four biological replicates were clustered into three major cell types (excitatory neurons, inhibitory neurons, and non-neuronal cells) using graph-based clustering, and then further sub-clustered within each category ( Figure 5F-5H and Figure 6C The unified manifold approximation (UMAP) plot, a nonlinear dimensionality reduction technique for visualizing similarities in cellular transcriptomes in two dimensions, showed consistent clustering of major cell types in 3,142 cells pooled from four biological replicates: 2,199 excitatory neurons, 324 inhibitory neurons, and 619 non-neuronal cells ( Fig. 5F ). Figure 5G The gene expression heatmaps for the 112 cell type markers shown in are aligned to each cell cluster and show clustering by inhibitory, excitatory, or non-neuronal cell type. The expression of each gene was z-scored for all genes in each cell. For representative cell-resolved spatial maps within and outside the neocortex, see Figure 5H ; For cell type codes, see Fig. 5F Clustering of excitatory and inhibitory subtypes ( Figure 5I-5N ) describes the UMAP map of representative genes ( Fig.5I and 5L ), Bar Chart( Figure 5J and 5M ) (mean ± SEM of gene expression for all cells in this cluster, each bar can be zoomed to the maximum mean expression of all clusters), and also describes excitability ( Figures 5I-5K ) and inhibitory ( Figure 5L-5N ) The in situ spatial distribution of neurons ( Figure 5K and 5N). The number of cells in each cluster is as follows: L2 / 3: 589; L4: 649; L5: 393; L6: 368; Pv neurons: 111; Vip neurons: 46; Sst neurons: 46; Npy neurons: 56. Inclusion of cells in clusters was based solely on amplicon representation in each cell without using spatial information; excitatory cell clusters were named based on the observed spatial stratification of the cluster, while inhibitory cell clusters were named based on the dominant cell type amplicon based on the high degree of segregation of amplicon markers.

[0191] Figure 6C A method for clustering and subclustering expression data for each cell is described: (1) the data are presented as a matrix of cells arranged by gene, which can be expressed as z-scored log-transformed counts; (2) principal component analysis (PCA) is applied to the matrix to reduce the matrix of cells arranged by factors; (3) the locations of the cells are indicated using visualization using the uniform manifold approximation (UMAP), a nonlinear dimensionality reduction technique for 2D visualization of high-dimensional data; (4) cells are clustered according to the PCA values ​​using graph clustering based on shared nearest neighbors; and (5) the expression values ​​of the cells corresponding to each cluster are taken and used again for subclustering.

[0192] Based on spatial correspondence with anatomical cortical layers and expression profiles of known layer-specific gene markers, well-defined excitatory neurons were divided into four major types, denoted (eL2 / 3, eL4, eL5, and eL6; Figures 5I-5K and Figure 7A-7B ). Fig. 7A The z-score expression matrix of excitatory cell types shown in Figure 1 lists the clustering of multiple differentially expressed genes by cell type. Genes expressed in cells in each cluster compared with cells in any other cluster were compared using a likelihood ratio test with a false discovery rate (FDR) adjusted P value threshold of 10 -10 and a log10 minimum fold change of 0.1 to select the genes displayed. Figure 7B UMAP visualization depicting the relative expression (normalized to the minimum and maximum values ​​across all excitatory cells) of multiple known layer-specific genes enriched in each cluster of cells, showing that most are enriched in specific excitatory subtypes. Figure 7C Describes Fig. 7A Expression matrix of selected suppressive cell types. Fig.7D Depicted is a UMAP visualization listing the relative expression of known interneuron marker genes, demonstrating that each is specifically enriched in inhibitory neuron subtypes.

[0193] Although the spatial organization of the four excitatory cell types was hierarchical, there was a high degree of intermixing between the different cell types within each layer. Inhibitory neurons were also clustered into four major types, represented by the dominant interneuron markers of each subtype (Vip, Sst, Npy, and Pvalb, Figure 5L-5N and Figure 7C-7D ); it was observed that Vip and Npy types were mainly distributed in the upper layers (L1-3), while Sst and Pvalb types were usually distributed in the lower layers (L4-6).

[0194] Non-neuronal cell types were also examined, including astrocytes, oligodendrocytes, endothelial cells, and smooth muscle cells ( Figures 8A-8C The number of major cell types shown here (12 in total) can be further subdivided (single-cell RNA sequencing can refine this to 40 or more subtypes, consistent with the marked heterogeneity of gene expression within each type; Figures 7A-7D ; Figures 8A-8C ). For UMAP visualization of four non-neuronal cell types, see Fig. 8A For a Z-scored expression matrix of non-neural cell types, see Figure 8B For genes expressed in cells in each cluster compared with cells in any other cluster, a likelihood ratio test was used with a false discovery rate (FDR)-adjusted P value threshold of 10 -10 and a log10 minimum fold change of 0.1 to select the genes displayed. Figure 8C UMAP visualization depicting the expression of marker genes for non-neuronal cell types (top differentially expressed genes for each cluster) per cell, indicating specificity for that cluster. Colors represent the relative expression of each gene across all non-neuronal cells (normalized to minimum and maximum values).

[0195] Using a targeted panel of 112 genes, all 12 major cell types were reliably detected at 600-800 cells per sample, with no batch effects and highly similar spatial patterns across four biological replicates ( Figures 9A-9G ). Figures 9A-9C Describes the sample replicates coded and then grouped by major clusters ( Fig. 9A ), excitatory sub-cluster ( Fig. 9B ) and inhibitory sub-clustering ( Fig. 9C ) were grouped by UMAP visualization. All cell types ( Fig.9D ), excitatory cell types ( Fig.9E ), suppressive cell types ( Fig.9F ) and non-neuronal cell types ( Figure 9G ) of the spatial map of the parallel measurements in the light and dark environments ( Figure 9D-9G , upper and lower figures). Example 6: Single-cell RNA sequencing

[0196] The quantitative power of STARmap at the single-cell level was evaluated to test differential gene expression analysis in molecularly defined cell types across experimental conditions. Visual stimulus-dependent gene expression patterns were assessed (enabled by 48 defined ARGs and single-cell in situ resolution). To further develop the single-cell RNA sequencing procedure, mouse brains were snap-frozen to minimize post-sacrifice processing time (less than 5 minutes) and without drug treatment to maximize preservation of the native transcriptional signature. Global induction of known immediate-early genes was observed in the primary visual cortex after 1 hour of light exposure (Fos, Egr1, and Egr2, Figures 10A-10D At single-cell resolution, the quantitative extent of ARG changes (fold change in expression) showed great diversity among various neuronal cell types ( Figures 10B-10C and Figures 11A-11C ). Fig. 10A The spatial expression pattern of a prototypic ARG called immediate early gene (IEG) in the visual cortex was verified. The mice were sacrificed in the dark or after 1 hour of light exposure. Fig. 10B and 10C Volcano plots depicting log fold changes in gene expression in inhibitory and excitatory cell types under light and dark conditions. Genes with significantly increased or decreased expression (false discovery rate adjusted P value < 0.05, Wilcoxon rank sum test) are marked in green, and genes with the most significant changes (P value < 0.05, fold change > 2) are marked in red. Many ARGs exhibit cell type specificity, suggesting an unexpected cell type-specific logic for excitation-transcription coupling. Fig. 10D Violin plots showing Egr2 expression by cell type are depicted. ****P < 0.0001, ns indicates not significant, Wilcoxon rank sum test; cell types marked in red with fold change greater than 2. In general, ARG expression programs in excitatory neurons of different layers were highly similar, whereas those in inhibitory cells showed distinct cell type-specific signatures ( Fig. 11B ); for example, Egr2 exhibits light-inducible activity in excitatory neurons ( Fig. 10D ), but not in inhibitory neurons, whereas Prok2 was upregulated in Vip inhibitory neurons ( Fig. 10C ).

[0197] Finally, because neural activity can trigger co-transcription of noncoding RNAs within ARG enhancers, examples of these enhancer RNAs were investigated (eRNAs 1-5 of the Fos gene); these unpolyadenylated transcripts are difficult to measure using current single-cell RNA sequencing. However, eRNA3 was identified as the most important and consistent ARG marker ( Fig. 11B ). Fig.11A Depicted are the correlations of 160 genes in biological replicates under dark / light conditions, showing that samples under the same conditions are more correlated than samples under different conditions; scale bar, Spearman R value. Fig. 11B Log-scaled expression data (counts per cell) of ARGs in inhibitory and excitatory neuronal subtypes are depicted. Genes with significantly increased expression in any cell type are highlighted in red. Fig. 11C Depicts a heatmap of neuronal subtype correlations based on the average expression correlation of all ARGs in a cluster that responded to light, showing that cells derived from inhibitory cell types are more correlated with other inhibitory cell types than with excitatory cell types, and vice versa. Note that the scale bar ranges from R = 0.8 to R = 1. Example 7: STARmap method for large tissue volumes

[0198] The 160-gene experiment was performed on brain slices no thicker than 1 cell body, so the ability of the STARmap method to capture the 3D structure of cells in tissue volumes has not been tested. STARmap was further developed to overcome the limitations of intact tissue volumes in terms of diffusion channels and imaging throughput, and to propose a strategy for linear readout of gene expression at cellular resolution to enable high-throughput molecular analysis in tissue volumes ( Fig.12 and Figure 13). Fig.12 The methods disclosed herein are described, including steps for sample size calibration, library preparation, imaging and sequencing, and data output. For thin tissue (z < 16 μm, monolayer cells), libraries were prepared using fresh frozen mouse brains, followed by cryostat sectioning, PFA fixation, permeabilization, hybridization, ligation and amplification, as well as hydrogel embedding and tissue clearing. Imaging and sequencing include single amplicon resolution; high NA oil immersion objectives; imaging of 200 cells per hour; SEDAL reactions with degenerate probes; and obtaining exponential reads of genes in cycles. Data output includes 3D amplicons and 2D cell typing ( Figures 1A-1E , Figures 5A-5N and Figures 10A-10D). For thicker tissues (z>100μm, with multiple cell layers), libraries were prepared from PFA-fixed mouse brains followed by vibratome sectioning, permeabilization and hybridization, hydrogel embedding and tissue clearing, and ligation and amplification. Imaging and sequencing include single-cell resolution; low NA water immersion objectives; imaging of 10,000 cells per hour; SEDAL with orthogonal probe reactions; and linear readout of genes in cycles. Data outputs include 3D cell typing ( Figures 13A-13I ).

[0199] Fig.13A Volumetric STAR mapping enabled by continuous SEDAL gene readout is described. The STARmap program ( Fig.12 , right) and cycle gene reads (4 genes per cycle), enabling rapid mapping of large volumes of tissue at single-cell resolution without oversampling of each amplicon. The specificity and penetration depth of large-volume STARmap were initially tested using Thy1::YFP mouse brain, where STARmap successfully detected YFP mRNA in tissue 150 μm thick and clearly co-localized YFP protein and mRNA at single-cell resolution ( Fig. 13B ) without labeling thousands of scattered neighboring cells. Validation shows specific STARMAP labeling of YFP-expressing neurons (derived from a transgenic Thy1::YFP mouse line) in 3D cortical layers. Scale bar 0.5 mm.

[0200] Spatial cell profiling of mouse primary visual cortex extended to more than 30,000 cells throughout six layers and the corpus callosum. Reads were segmented in 7 cycles of linear SEDAL sequencing using 28 genes including 23 cell type markers and 5 ARGs ( Figures 13C-13D and Figures 14A-14B ), K-means clustering (Methods) of the marker genes was applied to each cell type (11 cell types were recovered, which corresponded to the majority of cell types extracted by the 160-gene experiment, but only 28 genes were involved here). Fig. 13C Describes representative markers of major cell types obtained after multiple rounds of STARmap in the visual cortex ( Fig. 13C , left), layer-specific markers ( Fig. 13C , middle left), inhibitory markers ( Fig. 13C , middle right) and activity-regulated genes ( Fig. 13C , right).

[0201] 3D models of 11 cell types ( Figures 13E-13F) is consistent with the 160-gene thin-section tissue results, but provides accurate, quantitative analysis of the spatial distribution of cells with a much larger number of cells. Spatial histograms of excitatory, inhibitory, and non-neuronal cell types were generated using the same Fig.13D ( Fig.13E ) in the 2D maximum projections. Cells were counted in 5 μm bins and the density (cell count / μm) was plotted as a function of the distance between the corpus callosum (cc) and the pia mater, and the average was taken for the bars perpendicular to the cortical layers. The maximum projection cell positions were plotted according to Fig.13D ( Fig.13E , below). As the spatial histogram ( Fig.13E ) and related analysis diagram ( Fig. 14B ), the excitatory subtypes are distributed in layers, with the spatial density of each subtype gradually decreasing throughout the space until it is flush with the adjacent layer. Although the Vip subtype (mainly located in layers 2 / 3) and the Sst and Pvalb subtypes (in layers 4 and 5) show layer preferences, the inhibitory subtypes are more dispersed in comparison. Fig.14A The expression of each gene in the 3D maximum projection projected on the XY plane is depicted, which shows the spatial distribution of the extracted gene expression values ​​for each cell used for subsequent clustering. Across all genes, each gene was z-scored per cell. Fig. 14B Voxel-wise correlation coefficients between the distributions of each cell type binned into statistical bins on a 25 μm grid are depicted.

[0202] Non-neuronal cells were found mainly in layer 1 and the white matter. Fig.13D Depicts a cell-by-cell expression matrix of 28 genes derived from 32,845 single cells taken from material clustered into multiple excitatory, inhibitory, and non-neuronal cell types, with genes z-scored for each cell to normalize for the average difference in total signal between cells. Columns are sorted by sequencing run performed, in groups of 4. Fig.13F The spatial distribution of each cell type (excitatory, inhibitory, non-neuronal) and subtype is depicted in three dimensions. Each dot represents a single cell; spatial dimensions are in μm.

[0203] To find finer volume patterns, we analyzed the distribution of distances between individual cells of each sequencing-defined subtype and their nearest neighbors and unexpectedly found that the nearest neighbors of any inhibitory neuron tended to be its own subtype rather than excitatory neurons or other inhibitory subtypes ( Figure 13G ). For the average nearest neighbor distances between all excitatory cells (Excite) and each inhibitory cell type calculated in 3D, see Figure 13GFor self-comparison, the nearest neighbor was defined as the closest non-identical cell; persistent autocorrelations revealed the self-clustering of inhibitory subtypes. If inhibitory neurons were randomly distributed among more abundant excitatory neurons in a pure salt-and-pepper distribution, the distances between inhibitory neurons would be greater than the distances between inhibitory and excitatory neurons ( Fig.13H ).like Fig.13H As shown, the same distances were analyzed but with different (random) cell type markers. The actual intra-subtype distances for inhibitory neurons were much shorter (~15 μm, which is comparable to the size of a single neuron, indicating direct somatic apposition; Fig.13I ). Fig.13I Depicts the nearest neighbor distance calculated in 3D between each inhibitory cell of a certain type and any member of the same type (Inhib→Inhib, eg VIP→VIP) or any excitatory neuron (Inhib→Excite); ****P<0.0001, Wilcoxon rank-sum test. Fig.13I The self-clustering structure of the inhibitory isoform of the substance was revealed, which can only be accurately measured in 3D but not in 2D ( Figures 15A-15C ).exist Figures 13A-13I ( Fig.15A ), the nearest neighbor distances along the z direction between excitatory and different inhibitory cell types were calculated in 2D projections of 8 μm (thinner than one cell) slices within the same 3D volume shown in ). Fig.13D For the same cell type shown in , 2D nearest neighbor distances do not accurately estimate (overestimate) the corresponding 3D distances. This pattern may be functionally relevant; for example, in vivo imaging has shown that grouping of inhibitory neurons in the visual cortex can enhance visual responses. Fig. 15B An example of a 3D short-range clustering of inhibitory neurons is depicted, with a zoomed-in view as shown Fig. 13C shown. Fig. 15C Figure 3. Clusters of short-range inhibitory neurons observed in the primary visual cortex of transgenic mice (generated by Parv-IRES-Cre crosslinking with Ai14): Pvalb cells were labeled with tdtomato and all neuronal nuclei were immunostained with Alexa 647-conjugated anti-NeuN. Example 8: Scalability of STARmap

[0204] STARmap is suitable for longer sequencing lengths or more gene numbers; there is no inherent limit on the number of genes or RNA types, and STARmap can operate on them all quantitatively at the same time ( Figures 16A-16E ). Fig.16AThe detection of Actb mRNA in the presence of larger amounts of other RNAs is described to test potential dilution effects of the probe mixture and the physical capacity of the cells for SNAIL DNA amplicons. The SNAIL probe for Actb was designed with an orthogonal DNA sequence for detection and was incorporated into a mixture containing 0( Fig.16A )、100( Fig. 16B ) or 1,000( Fig. 16C ) other genes. If the SNAIL probes do not work as efficiently as individual probes in a mixed environment, or if there is not enough space for rolling circle amplification, the insertion of the Actb gene will result in fewer amplicons and / or reduced intensity of each amplicon. Fluorescence image acquired in mouse visual cortex; green: Alexa 546 channel for Actb amplicon; red: Alexa 647 channel for all other genes; blue: DAPI staining of cell nuclei. Figures 16A-16C For quantitative information, see Fig.16D Box plots show that any effects of dilution and cellular spatial confinement are negligible at least on a 1,000-gene scale. Boxes indicate first and third quartiles; center line indicates median; whiskers indicate 5% and 95% data points; n indicates number of Actb amplicons in the entire 228x228x2μm imaging volume; y-axis: absolute fluorescence intensity. Fig.16E Experimental and theoretical estimates of STARmap's scalability are described. Encoding suggests that a 5-nt codon can encode 1,024 genes; SNAIL probes have 35-nt of coding space in addition to the RNA complement. SEDAL requires 17-nt as a sequencing unit (11-nt docking region for the read probe, plus the 5-nt codon and 1-nt flanking base), so SNAIL probes can accommodate two such units, allowing for the presence of 4 10 (10 6 ) codons; the upper limit is closer to 10 if other sequencing methods are used for longer reads (e.g., SOLiD, 18-nt for primer binding and 17-nt for coding) 11 A physical capacity of up to 1000 genes has been demonstrated in mammalian neurons; since the physical size of DNA amplicons determined by AFM and TEM is approximately 100-200 nm, the maximum capacity is estimated to be 10 per cell, assuming a cell diameter of approximately 15 μm and using a close-packed model (spatial efficiency of 74%). 6amplicons. Optical volume was validated for mouse hippocampal cell cultures using a 1,020-gene experiment and visual cortex experiments; amplicons / cell are those that were successfully aligned in all six rounds of sequencing. When imaged using confocal microscopy, the average diameter of DNA amplicons is 400-600nm; using the same model used in the physical limit experiments, the maximum volume is 2x10 per cell. 4 amplicons. The experimental data for 1,020 genes are also close to this limit. Without being bound by any scientific theory, the numerical differences between cell culture and tissue sections may be attributed to the following considerations: (1) whole cells were imaged in cell culture, while cell fragments were imaged in tissue sections (8 μm, <1 cell thickness); (2) hippocampal cell cultures are less differentiated than adult mouse brain, so there is a greater difference in the RNA contained in each cell (more genes); and (3) in contrast to the dense 3D packing of cells in brain tissue, cells in vitro are more dispersed in the xy plane and gradually thin in the z direction, and images in the xy plane show higher optical resolution than in the z direction (voxel size is 78x78x250 μm). Example 9: Neural networks identified in STARmap160-gene experiments and published single-cell RNA sequencing results Metatype Relevance

[0205] Fig.17 Pearson correlations of mean gene expression for all genes in the identified STARmap excitatory and inhibitory clusters and the corresponding clusters identified by single-cell RNA-seq acquired from the Allen Brain Institute are depicted. See, e.g., Lein et al., (2007). For single-cell RNA-seq data, expression of all subtypes within the major classes (e.g., L2 / 3) was averaged, and correlations were calculated using only genes in common between the single-cell RNA-seq and 160-gene V1 experiments; scale bar 0-0.6, Pearson correlation coefficient. Example 10: Gene expression analysis of cell type sub-clusters in the medial prefrontal cortex (mPFC) using STARmap analyze

[0206] Fig.18A Depicted is the UMAP visualization of excitatory subclusters. Fig.18B Differentially expressed genes for each excitatory subcluster are depicted. Fig.18C UMAP visualization of suppressive subclusters is depicted. Fig.18D The differentially expressed genes of each suppressive subcluster are depicted. Fig.18E Depicted is the UMAP visualization of the non-neural subclusters. Fig.18F Differentially expressed genes for each non-neural subcluster are depicted.

[0207] Figures 19A-19C Describes the excitatory subclusters ( Fig.19A ), inhibitory sub-clustering ( Fig.19B ) and non-neuronal subclusters ( Fig.19C ) spatial plot of four biological replicates. Example 11: Gene expression analysis of 1020 genes by cell type sub-clustering in mouse hippocampal cell culture

[0208] Figures 20A-20C Described is the analysis of 1020 genes in mouse hippocampal cell cultures in 6 rounds of sequencing using the methods described herein. Fig. 20A The raw fluorescence images of the four fluorescence channels merged from the first round are depicted. Fig. 20B Examples of cell type markers are depicted. Neuronal gene markers (Scna) are appropriately separated from non-neuronal gene markers (Mt1), and the distribution of neuronal subtype markers (Reln, Sst) is also very clear. Fig. 20C Statistical analysis of amplicons and genes per cell is depicted, with an imaged area of ​​270x270 μm.

[0209] Figures 21A-21C Additional gene expression information obtained by manipulating 1020 genes in mouse primary visual cortex using the methods described herein is described. Fig.21A UMAP visualization of excitatory subclusters is depicted ( Fig.21A , left) and differentially expressed genes in each excitatory subcluster ( Fig.21A , right). Fig. 21B Depicts UMAP visualization of suppressive subclusters ( Fig. 21B , left) and differentially expressed genes in each inhibitory subcluster ( Fig. 21B , right). Fig. 21C Describes the UMAP visualization of non-neural subclusters ( Fig. 21C , left) and the differentially expressed genes of each non-neural sub-cluster ( Fig. 21C , right).

[0210] Figures 22A-22D Describes the reproducibility determination and cross-method comparison of measurements of 1020 genes in the mouse primary visual layer using STARmap. Fig.22A Depicted is the correlation of reads per gene between two 1,020-gene replicates in the visual cortex. Fig. 22B Describes the histogram of detected reads for each cell ( Fig. 22B , left) and detected genes per cell ( Fig. 22B , right). Fig. 22C Describes spatial maps of cell types in additional replicates of the 1,020-gene visual cortex experiment. Fig.22DDepicted are Pearson correlations of mean gene expression for all genes in the identified STARmap 1,020-gene cluster and the corresponding cluster identified by single-cell RNA-seq obtained from the Allen Brain Institute. Example 12: STARmap for thin and thick tissue sections

[0211] Fig.23A Experimental flow chart describing STARmap applicable to thin and thick tissues. Fig. 23B The preparation of modified primer probes for large volume experiments is shown: DNA probes with 5' amine modifications are ordered, mixed, and converted to polymerizable moieties by AA-NHS. Fig.23C The experimental durations for different experimental designs with various numbers of genes are shown. Fig.23D A comparison of STARmap with other single-cell methods in terms of RNA species, spatial resolution, and throughput is shown. Single-cell RNA sequencing can be combined with recently developed spatial transcriptomic methods to obtain regional spatial resolution (100 μm).

[0212] Although described with reference to specific embodiments of the present invention, it will be appreciated by those skilled in the art that various changes may be made and may be replaced with equivalents without departing from the true spirit and scope of the present invention. In addition, various modifications may be made to adapt a particular situation, material, composition of matter, process, or one or more operating steps to the purpose, spirit, and scope of the present invention. All such modifications are intended to be covered by the scope of the appended claims. References 1. N. CROSETTO, M. BIENKO, A. VAN OUDENAARDEN, Spatially resolved transcriptomics and beyond, Nature Reviews Genetics, 16, 57-66. 2. E. LEIN, LEBORM, S. LINNARSSON, Prospects of applying spatial transcriptomics to neuroscience in the era of molecular cell typing, Science, 358, 64-69 (2017). 3. E. LUBECK, L. 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RYTAM, LJSMITH, MSSHOICHET, Modification of cell microenvironment using photo- and enzymatic hydrogels: towards a biomimetic 3D cell culture model, Chemical Research Reviews, 50, 703-713 (2017). 10. K. CHUNG et al., Structural and molecular interrogation of intact biological systems, Nature, 497, 332-337 (2013). 11. ELSYLWESTRAK, P. RAJASETHUPATHY, MAWRIGHT, A. JAFFE, K. DEISSEROTH. Multiplexed transcriptional profiling of intact tissues at cellular resolution. Cell, 164, 792-804 (2016). 12. S. SHAL et al., Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and removal, Development, 143, 2862-2867 (2016). 13. J. R. M. Offitt et al., High-performance multiplex fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing, Proceedings of the National Academy of Sciences of the United States of America, 113, 14456-14461 (2016). 14. F. CHEN et al., Nanoscale imaging of RNA using expansion microscopy, Nature Methods, 13, 679-684 (2016). 15. J. SHENDURE et al., Accurate multiplex polymerase clone sequencing of evolved bacterial genomes, Science, 309, 1728-1732 (2005). 16. JHLEE et al., Fluorescent in situ sequencing of RNA (FISSEQ) for gene expression profiling in intact cells and tissues, Nature Experiments, 10, 442-458 (2015). 17. R. DRMANAC et al., Human genome sequencing using melt base reads on self-assembled DNA nanoarrays, Science, 327, 78-81 (2010). 18. LLGLICKFELD, RCREID, MLANDERMANN. A mouse model with higher-order visual cortical functions. Current Opinion in Neurobiology 24, 28-33 (2014). 19. B. Tasic et al., Cellular classification in adult mouse cortex revealed by single-cell transcriptomics, Nature Neuroscience, 19, 335–346 (2016). 20. A. ZEISEL et al., Cell types in mouse cortex and hippocampus revealed by single-cell RNA-seq, Science, 347, 1138-1142 (2015). 21. 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Claims

1. A method for identifying nucleic acids in cells in a tissue, comprising: (a) contacting the tissue with at least one pair of oligonucleotides, wherein the contacting occurs under conditions that allow specific hybridization of the at least one pair of oligonucleotides to the nucleic acid to occur, the at least one pair of oligonucleotides comprising: (i) a first oligonucleotide, wherein the first oligonucleotide comprises a first region of complementarity, a second region of complementarity and a third region of complementarity; and (ii) a second oligonucleotide, wherein the second oligonucleotide comprises a fourth region of complementarity, a fifth region of complementarity, and a sixth region of complementarity; and wherein the first complementary region is complementary to a first portion of the nucleic acid, the second complementary region is complementary to the fourth complementary region, the third complementary region is complementary to the sixth complementary region, the fifth complementary region is complementary to a second portion of the nucleic acid, and the first complementary region and the fifth complementary region hybridize to the nucleic acid; (b) performing an amplification reaction on the nucleic acid having the at least one pair of oligonucleotides hybridized thereto to produce one or more amplicons, wherein the one or more amplicons are embedded in the hydrogel; and (c) imaging the one or more hydrogel-embedded amplicons to identify the nucleic acid.

2. The method according to claim 1, further comprising, after (b), removing the plurality of cellular components in the tissue after step (b).

3. The method according to claim 2, wherein: The cellular components include lipids, proteins, or a combination of both.

4. The method according to any one of claims 1 to 3, wherein: The at least one pair of oligonucleotides is denatured by heating prior to contacting the tissue.

5. The method according to any one of claims 1 to 4, wherein: The cells are present in a cell population.

6. The method according to claim 5, wherein: The cell population includes a plurality of cell types.

7. The method according to any one of claims 1 to 6, wherein: The tissues were fixed and permeabilized with a single solution.

8. The method according to any one of claims 1 to 7, wherein: The nucleic acid is RNA.

9. The method according to any one of claims 1 to 8, wherein: The nucleic acid is messenger RNA.

10. The method according to any one of claims 1 to 9, wherein: The nucleic acid is DNA.

11. The method according to any one of claims 1 to 10, wherein: The second oligonucleotide is provided in the form of a closed nucleic acid circle.

12. The method according to any one of claims 1 to 11, wherein: (a) the second oligonucleotide comprises a padlock probe, (b) the length of the first complementary region of the first oligonucleotide is 19 to 25 nucleotides, (c) the second complementary region of the first oligonucleotide is 3 to 10 nucleotides in length, (d) the third region of complementarity of the first oligonucleotide is 3 to 10 nucleotides in length, (e) the fourth region of complementarity of the second oligonucleotide is 3 to 10 nucleotides in length, (f) the length of the fifth region of complementarity of the second oligonucleotide is 19 to 25 nucleotides, (g) the length of the sixth region of complementarity of the second oligonucleotide is 3 to 10 nucleotides, (h) the fourth region of complementarity comprises the 5' end of the second oligonucleotide, (i) the sixth region of complementarity comprises the 3' end of the second oligonucleotide, and (j) the fourth region of complementarity of the second oligonucleotide is adjacent to the sixth region of complementarity of the second oligonucleotide, or a combination thereof.

13. The method according to any one of claims 1 to 12, wherein: (k) a third oligonucleotide is configured to decode a base, and (l) The fourth oligonucleotide is configured to convert the decoded base into a signal.

14. The method according to any one of claims 1 to 13, wherein: The imaging comprises imaging the one or more hydrogel-embedded amplicons using a microscopy technique selected from the group consisting of: Confocal microscopy, two-photon microscopy, light-field microscopy, intact tissue expansion microscopy, super-resolution microscopy, and thin-section microscopy.

15. The method according to claim 14, wherein: The thin-section microscopy technique is CLARITYTM optimized thin-section microscopy (COLM).

16. The method according to any one of claims 1 to 15, wherein: The thickness of the tissue is 5 microns to 20 microns.

17. The method according to any one of claims 1 to 16, wherein: The tissue is a thick slice with a thickness of 5 micrometers to 200 micrometers.

18. The method of any one of claims 1-17, further comprising adding a ligase to ligate the first end of the second oligonucleotide to the second end of the second oligonucleotide, thereby generating a closed nucleic acid circle.

19. The method according to claim 18, wherein: The ligase is DNA ligase.

20. The method according to any one of claims 1 to 19, wherein: The amplification reaction comprises a rolling circle amplification, wherein the second oligonucleotide is a template and the first oligonucleotide is a primer for a polymerase to generate one or more amplicons.

21. The method according to any one of claims 1 to 20, wherein: After the amplification reaction in (b), the one or more amplicons are embedded in a hydrogel.

22. The method according to any one of claims 1 to 21, wherein: One or more amplicons are embedded in the hydrogel by copolymerizing the one or more amplicons with acrylamide.

23. The method of any one of claims 1-21, wherein the tissue is embedded in a hydrogel prior to (b).

24. The method according to any one of claims 1 to 21, wherein: The first portion and the second portion of the nucleic acid are separated by one or more nucleotides.

25. The method according to any one of claims 1 to 21, wherein: The first complementarity region is adjacent to the fifth complementarity region.

26. A system comprising: A device comprises an imaging chamber, a fluid device and a processor unit, wherein the processor unit is configured to implement the method according to any one of claims 1-25.

27. The system of claim 26, wherein: The fluid device includes a pump.

28. A method for screening a candidate agent to determine whether the candidate agent can modulate gene expression of a nucleic acid in a cell within an intact tissue, the method comprising: (a) performing the method according to any one of claims 1 to 25 to determine the sequence of nucleic acids in cells in a tissue, and (b) detecting the gene expression level of the target nucleic acid, wherein a change in the expression level of the target nucleic acid in the presence of the at least one candidate agent relative to the expression level of the target nucleic acid in the absence of the at least one candidate agent indicates that the at least one candidate agent regulates gene expression of nucleic acids in cells within intact tissue.

29. The method of claim 28, wherein the detecting comprises determining a signal.

30. The method of claim 29, wherein the signal is a fluorescent signal.

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