Methods for determining in-situ gene sequences

Abstract

To provide devices, methods, and systems for in situ gene sequencing of a target nucleic acid in a cell in an intact tissue.SOLUTION: Methods of screening a candidate agent to determine whether the candidate agent modulates gene expression of a nucleic acid in a cell in an intact tissue are also provided herein. The present disclosure provides a method for in situ gene sequencing of a target nucleic acid in a cell in an intact tissue. In some cases, a pair of primers are denatured by heating before contact with a sample. In some cases, the cell is present in a population. In some cases, the population of cells includes a plurality of cell types.SELECTED DRAWING: None

Description

[Technical Field] 【0001】 cross reference This application claims the interests of U.S. Provisional Patent Application No. 62 / 655,052 filed on 9 April 2018, U.S. Provisional Patent Application No. 62 / 687,490 filed on 20 June 2018, and U.S. Provisional Patent Application No. 62 / 808,159 filed on 20 February 2019, which are incorporated herein by reference in their entirety. [Background technology] 【0002】 In biological tissues, functional diversity arises, in part, from morphological diversity through the complexity of cell-specific gene expression, which defines the unique three-dimensional molecular biostructure and cellular properties of each tissue. The emergence of in-situ transcriptional tools for spatial mapping of gene expression at sub-cell resolution may be applicable to exploring these tissue structure-function relationships, including both multiplexed in-situ RNA hybridization and in-situ RNA sequencing. Current in-situ sequencing approaches face the challenge of performing enzymatic reactions in high-density, complex tissue environments and currently suffer from low efficiency. However, the potential value of such in-tissue sequencing can be enormous, and compared to multiplexing / readout based on hybridization that utilizes numerous polynucleotide probes to encode genetic identity, sequencing operates at single-nucleotide resolution and therefore, in essence, provides far greater information. However, current sequencing methods are not yet applicable to the 3D volume of intact tissue due to fundamental limitations in the sensitivity, fidelity, and scalability required for throughput in tissues such as the mammalian brain. For example, the mammalian brain is composed of a complex tapestry of cell types, along with the diversity essential for function arising from both differential gene expression and circuit-specific biostructure. Obtaining high-resolution gene expression information while preserving 3D positional biostructure at cellular resolution has been challenging and has limited our integrated understanding of brain structure and function. This disclosure addresses the above issues and offers relevant advantages. [Overview of the project] [Means for solving the problem] 【0003】 Devices, methods, and systems for in-situ gene sequencing of target nucleic acids in cells within intact tissue are provided herein. Methods for screening candidate drugs to determine whether a candidate drug modulates gene expression of nucleic acids in cells within intact tissue are also provided herein. 【0004】 This disclosure provides a method for in situ gene sequencing of a target nucleic acid in cells in intact tissue, the method comprising (a) contacting immobilized and permeabilized intact tissue with at least a pair of oligonucleotide primers under conditions that enable specific hybridization, wherein the primer pair comprises a first oligonucleotide and a second oligonucleotide, each of which comprises a first complementary region, a second complementary region, and a third complementary region, and the second oligonucleotide is The first complementary region of the first oligonucleotide is complementary to the first portion of the target nucleic acid, the second complementary region of the first oligonucleotide is complementary to the first complementary region of the second oligonucleotide, the third complementary region of the first oligonucleotide is complementary to the third complementary region of the second oligonucleotide, the second complementary region of the second oligonucleotide is complementary to the second portion of the target nucleic acid, and the first complementary region of the first oligonucleotide is adjacent to the second complementary region of the second oligonucleotide. (b) bringing into contact with (c) adding ligase to ligate the second oligonucleotide and generate a closed nucleic acid ring, (d) performing rolling circle amplification in the presence of nucleic acid molecules, using the second oligonucleotide as a template and the first oligonucleotide as a primer for polymerase to form one or more amplicons, (e) embedding one or more amplicons in the presence of hydrogel subunits to form amplicons embedded in one or more hydrogels, (f) bringing one or more hydrogel-embedded amplicons having a barcode sequence into contact with a pair of primers under conditions that allow ligation, the pair of primers comprising a third oligonucleotide and a fourth oligonucleotide, and ligation occurring only when both the third and fourth oligonucleotides ligate to the same amplicon, (g) repeating step (e) many times, and (f) imaging the amplicons embedded in one or more hydrogels.This includes determining the in-situ gene sequence of a target nucleic acid in cells within intact tissue. In some cases, a pair of primers are denatured by heating before contact with the sample. In some cases, the cells are present in a population. In some cases, the cell population includes multiple cell types. 【0005】 The disclosure also provides a method for screening candidate drugs to determine whether a candidate drug modulates gene expression of nucleic acids in cells within intact tissue, the method comprising (a) contacting immobilized and permeabilized intact tissue with at least a pair of oligonucleotide primers under conditions that allow specific hybridization, wherein the primer pair comprises a first oligonucleotide and a second oligonucleotide, each of the first and second oligonucleotides comprising a first complementary region, a second complementary region, and a third The first oligonucleotide comprises a complementary region, the second oligonucleotide further comprises a barcode sequence, the first complementary region of the first oligonucleotide is complementary to the first portion of the target nucleic acid, the second complementary region of the first oligonucleotide is complementary to the first complementary region of the second oligonucleotide, the third complementary region of the first oligonucleotide is complementary to the third complementary region of the second oligonucleotide, the second complementary region of the second oligonucleotide is complementary to the second portion of the target nucleic acid, and the first complementary region of the first oligonucleotide is, (b) contacting the second complementary region of the second oligonucleotide adjacent to it, (c) adding a ligase to ligate the second oligonucleotide and generate a closed nucleic acid ring, (d) performing rolling circle amplification in the presence of nucleic acid molecules, using the second oligonucleotide as a template and the first oligonucleotide as a primer for polymerase to form one or more amplicons, (e) embedding one or more amplicons in the presence of a hydrogel subunit to form amplicons embedded in one or more hydrogels, (e) contacting the amplicons embedded in one or more hydrogels having a barcode sequence with a pair of primers under conditions that allow ligation, the pair of primers comprising a third oligonucleotide and a fourth oligonucleotide, and ligation occurring only when both the third oligonucleotide and the fourth oligonucleotide ligate to the same amplicon, and (f) repeating step (e) many times.(g) imaging amplicons embedded in one or more hydrogels to determine the in-situ gene sequence of a target nucleic acid in cells within intact tissue; and (h) detecting the level of gene expression of the target nucleic acid, wherein the change in the level of expression of the target nucleic acid in the presence of at least one candidate drug compared to the level of expression of the target nucleic acid in the absence of at least one candidate drug indicates that at least one candidate drug modulates gene expression of the nucleic acid in cells within intact tissue. In some cases, a pair of primers are denatured by heating before contact with the sample. In some cases, the cells are present in a population of cells. In some cases, the population of cells includes multiple cell types. 【0006】 Devices used in accordance with the methods described herein are also provided. In some embodiments, a fluid system is provided for automating the methods described herein to enable continuous operation. In some embodiments, the system comprises a fluid device and a processor configured to perform the methods described herein. In a particular embodiment, the following items are provided, for example: (Item 1) A method for determining the in-situ gene sequence of a target nucleic acid in cells within intact tissue, (a) Contacting immobilized and permeabilized intact tissue with at least one pair of oligonucleotide primers under conditions that enable specific hybridization, The primer pair comprises a first oligonucleotide and a second oligonucleotide, Each of the first oligonucleotide and the second oligonucleotide comprises a first complementary region, a second complementary region, and a third complementary region, and the second oligonucleotide further comprises a barcode sequence. The first complementary region of the first oligonucleotide is complementary to the first portion of the target nucleic acid, the second complementary region of the first oligonucleotide is complementary to the first complementary region of the second oligonucleotide, the third complementary region of the first oligonucleotide is complementary to the third complementary region of the second oligonucleotide, the second complementary region of the second oligonucleotide is complementary to the second portion of the target nucleic acid, and the first complementary region of the first oligonucleotide is adjacent to the second complementary region of the second oligonucleotide, thus creating contact between them. (b) Adding ligase to ligate the second oligonucleotide and generate a closed nucleic acid ring, (c) Performing rolling circle amplification in the presence of nucleic acid molecules, the second The process includes forming one or more amplicons using an oligonucleotide as a template and the first oligonucleotide as a primer for a polymerase, (d) Embedding one or more amplicons in the presence of hydrogel subunits to form amplicons embedded in one or more hydrogels, (e) Contacting the amplicons embedded in one or more hydrogels having the barcode sequence with a pair of primers under conditions that enable ligation, wherein the pair of primers comprises a third oligonucleotide and a fourth oligonucleotide, and the ligation occurs only when both the third oligonucleotide and the fourth oligonucleotide ligate to the same amplicon. (f) Repeat step (e) many times, (g) A method comprising imaging an amplicon embedded in one or more hydrogels to determine the in situ gene sequence of the target nucleic acid in the cells within the intact tissue. (Item 2) The method according to item 1, wherein the pair of primers is denatured by heating before contacting with the sample. (Item 3) The method according to item 1 or 2, wherein the cell exists within a population of cells. (Item 4) The method according to item 3, wherein the population of cells contains a plurality of cell types. (Item 5) The method according to any one of items 1 to 4, wherein contacting the fixed and permeabilized intact tissue comprises hybridizing the pair of primers to the same target nucleic acid. (Item 6) The method according to any one of items 1 to 5, wherein the target nucleic acid is RNA. (Item 7) The method according to item 6, wherein the RNA is mRNA. (Item 8) The method according to any one of items 1 to 5, wherein the target nucleic acid is DNA. (Item 9) The method according to any one of items 1 to 8, wherein the second oligonucleotide comprises a padlock probe. (Item 10) The method according to any one of items 1 to 9, wherein the first complementary region of the first oligonucleotide has a length of 19 to 25 nucleotides. (Item 11) The method according to any one of items 1 to 10, wherein the second complementary region of the first oligonucleotide has a length of 6 nucleotides. (Item 12) The method according to any one of items 1 to 11, wherein the third complementary region of the first oligonucleotide has a length of 6 nucleotides. (Item 13) The method according to any one of items 1 to 12, wherein the first complementary region of the second oligonucleotide has a length of 6 nucleotides. (Item 14) The method according to any one of items 1 to 13, wherein the second complementary region of the second oligonucleotide has a length of 19 to 25 nucleotides. (Item 15) The method according to any one of items 1 to 14, wherein the third complementary region of the second oligonucleotide has a length of six nucleotides. (Item 16) The method according to any one of items 1 to 15, wherein the first complementary region of the second oligonucleotide comprises the 5' end of the second oligonucleotide. (Item 17) The method according to any one of items 1 to 16, wherein the third complementary region of the second oligonucleotide comprises the 3' end of the second oligonucleotide. (Item 18) The method according to any one of items 1 to 17, wherein the first complementary region of the second oligonucleotide is adjacent to the third complementary region of the second oligonucleotide. (Item 19) The method according to any one of items 1 to 18, wherein the barcode sequence of the second oligonucleotide provides barcode information for identifying the target nucleic acid. (Item 20) The method according to any one of items 1 to 19, wherein contacting the immobilized and permeabilized intact tissue comprises hybridizing a plurality of oligonucleotide primers having specificity for different target nucleic acids. (Item 21) The method according to item 1, wherein the second oligonucleotide is provided as a closed nucleic acid ring, and the step of adding ligase is omitted. (Item 22) Melting temperature of oligonucleotides (T m ) a method according to any of items 1 to 21, selected to minimize ligation in the solution. (Item 23) The method according to any one of items 1 to 22, wherein the addition of the ligase includes the addition of a DNA ligase. (Item 24) The method according to any one of items 1 to 23, wherein the nucleic acid molecule comprises an amine-modified nucleotide. (Item 25) The method according to item 24, wherein the amine-modified nucleotide comprises a partial modification of N-hydroxysuccinimide acrylate. (Item 26) The method according to any one of items 1 to 25, wherein the embedding comprises copolymerizing one or more amplicons with acrylamide. (Item 27) The method according to any one of items 1 to 26, wherein the embedding comprises clarifying the amplicons embedded in the one or more hydrogels, and the target nucleic acid is substantially retained in the amplicons embedded in the one or more hydrogels. (Item 28) The method according to item 27, wherein the clarification includes substantially removing a plurality of cellular components from the amplicons embedded in the one or more hydrogels. (Item 29) The method according to item 27 or 28, wherein the clarification includes substantially removing lipids from the amplicons embedded in the one or more hydrogels. (Item 30) The method according to any one of items 1 to 29, wherein the third oligonucleotide is configured to decode a base. (Item 31) The method according to any one of items 1 to 30, wherein the fourth oligonucleotide is configured to convert the decoded base into a signal. (Item 32) The method according to item 31, wherein the signal is a fluorescent signal. (Item 33) The method according to any one of items 1 to 32, wherein the contact of the amplicons embedded in one or more hydrogels is performed to eliminate the accumulation of errors as sequencing progresses. (Item 34) The method according to any one of items 1 to 33, wherein the imaging includes imaging the amplicons embedded in one or more hydrogels using confocal microscopy, two-photon microscopy, bright-field microscopy, intact tissue expansion microscopy, and / or CLARLITY® optimized light sheet microscopy (COLM). (Item 35) The method according to any one of items 1 to 34, wherein the intact tissue is a thin section. (Item 36) The method according to item 35, wherein the intact tissue has a thickness of 5 to 20 μm. (Item 37) The method according to item 35 or 36, wherein the contact of the amplicons embedded in one or more hydrogels occurs four or more times. (Item 38) The method according to item 35 or 36, wherein the contact of the amplicons embedded in one or more hydrogels occurs five or more times. (Item 39) The method according to any one of items 1 to 34, wherein the intact tissue is a thick section. (Item 40) The method according to item 39, wherein the intact tissue has a thickness of 50 to 200 μm. (Item 41) The method according to item 39 or 40, wherein the amplicons embedded in one or more hydrogels are brought into contact six or more times. (Item 42) The method according to item 39 or 40, wherein the amplicons embedded in one or more hydrogels are brought into contact seven or more times. (Item 43) A method for screening candidate drugs and determining whether the candidate drugs regulate the gene expression of nucleic acids in cells within intact tissue, (a) Contacting immobilized and permeabilized intact tissue with at least one pair of oligonucleotide primers under conditions that enable specific hybridization, The primer pair comprises a first oligonucleotide and a second oligonucleotide, Each of the first oligonucleotide and the second oligonucleotide comprises a first complementary region, a second complementary region, and a third complementary region, and the second oligonucleotide further comprises a barcode sequence. The first complementary region of the first oligonucleotide is complementary to the first portion of the target nucleic acid, the second complementary region of the first oligonucleotide is complementary to the first complementary region of the second oligonucleotide, the third complementary region of the first oligonucleotide is complementary to the third complementary region of the second oligonucleotide, and the second complementary region of the second oligonucleotide is complementary to the second portion of the target nucleic acid. They are partially complementary, and the first complementary region of the first oligonucleotide is adjacent to the second complementary region of the second oligonucleotide, in contact with it. (b) Adding ligase to ligate the second oligonucleotide and generate a closed nucleic acid ring, (c) Performing rolling circle amplification in the presence of nucleic acid molecules, comprising forming one or more amplicons using the second oligonucleotide as a template and the first oligonucleotide as a primer for polymerase, (d) Embedding one or more amplicons in the presence of hydrogel subunits to form amplicons embedded in one or more hydrogels, (e) Contacting the amplicons embedded in one or more hydrogels having the barcode sequence with a pair of primers under conditions that enable ligation, wherein the pair of primers comprises a third oligonucleotide and a fourth oligonucleotide, and the ligation occurs only when both the third oligonucleotide and the fourth oligonucleotide ligate to the same amplicon. (f) Repeat step (e) many times, (g) Image the amplicons embedded in one or more hydrogels to determine the in-situ gene sequence of the target nucleic acid in the cells within the intact tissue, (h) A method comprising detecting the level of gene expression of the target nucleic acid, wherein the change in the level of expression of the target nucleic acid in the presence of the at least one candidate drug compared to the level of expression of the target nucleic acid in the absence of the at least one candidate drug indicates that the at least one candidate drug modulates gene expression of the nucleic acid in the cells in the intact tissue. (Item 44) The method according to item 43, wherein the pair of primers are denatured by heating before contact with the sample. (Item 45) The method according to item 43 or 44, wherein the cells are present within a population of cells. (Item 46) The method according to item 45, wherein the population of cells comprises multiple cell types. (Item 47) The method according to any one of items 43 to 46, wherein contacting the immobilized and permeabilized intact tissue comprises hybridizing the pair of primers to the same target nucleic acid. (Item 48) The method according to any one of items 43 to 47, wherein the target nucleic acid is RNA. (Item 49) The method described in item 48, wherein the RNA is mRNA. (Item 50) The method according to any one of items 43 to 47, wherein the target nucleic acid is DNA. (Item 51) The method according to any one of items 43 to 50, wherein the second oligonucleotide comprises a padlock probe. (Item 52) The method according to any one of items 43 to 51, wherein the first complementary region of the first oligonucleotide has a length of 19 to 25 nucleotides. (Item 53) The method according to any one of items 43 to 52, wherein the second complementary region of the first oligonucleotide has a length of six nucleotides. (Item 54) The method according to any one of items 43 to 53, wherein the third complementary region of the first oligonucleotide has a length of six nucleotides. (Item 55) The method according to any one of items 43 to 54, wherein the first complementary region of the second oligonucleotide has a length of six nucleotides. (Item 56) The method according to any one of items 43 to 55, wherein the second complementary region of the second oligonucleotide has a length of 19 to 25 nucleotides. (Item 57) The method according to any one of items 43 to 56, wherein the third complementary region of the second oligonucleotide has a length of six nucleotides. (Item 58) The method according to any one of items 43 to 57, wherein the first complementary region of the second oligonucleotide comprises the 5' end of the second oligonucleotide. (Item 59) The method according to any one of items 43 to 58, wherein the third complementary region of the second oligonucleotide comprises the 3' end of the second oligonucleotide. (Item 60) The method according to any one of items 43 to 59, wherein the first complementary region of the second oligonucleotide is adjacent to the third complementary region of the second oligonucleotide. (Item 61) The method according to any one of items 43 to 60, wherein the barcode sequence of the second oligonucleotide provides barcode information for identifying the target nucleic acid. (Item 62) The method according to any one of items 43 to 61, wherein contacting the immobilized and permeabilized intact tissue comprises hybridizing a plurality of oligonucleotide primers having specificity for different target nucleic acids. (Item 63) The method according to item 43, wherein the second oligonucleotide is provided as a closed nucleic acid ring, and the step of adding ligase is omitted. (Item 64) Melting temperature of oligonucleotides (T m ) the method described in any of items 43-63, selected to minimize ligation in the solution. (Item 65) The method according to any one of items 43 to 64, wherein the addition of the ligase includes the addition of a DNA ligase. (Item 66) The method according to any one of items 43 to 65, wherein the nucleic acid molecule comprises an amine-modified nucleotide. (Item 67) The method according to item 66, wherein the amine-modified nucleotide comprises a partial modification of N-hydroxysuccinimide acrylate. (Item 68) The method according to any one of items 43 to 67, wherein the embedding comprises copolymerizing one or more amplicons with acrylamide. (Item 69) The method according to any one of items 43 to 68, wherein the embedding comprises clarifying the amplicons embedded in the one or more hydrogels, and the target nucleic acid is substantially retained in the amplicons embedded in the one or more hydrogels. (Item 70) The method according to item 69, wherein the clarification includes substantially removing a plurality of cellular components from the amplicons embedded in the one or more hydrogels. (Item 71) The method according to item 69 or 70, wherein the clarification includes substantially removing lipids from the amplicons embedded in the one or more hydrogels. (Item 72) The method according to any one of items 43 to 71, wherein the third oligonucleotide is configured to decode the base. (Item 73) The method according to any one of items 43 to 72, wherein the fourth oligonucleotide is configured to convert the decoded base into a signal. (Item 74) The method according to item 73, wherein the signal is a fluorescent signal. (Item 75) The method according to any one of items 43 to 74, wherein the contact of the amplicons embedded in one or more hydrogels is performed to eliminate the accumulation of errors as sequencing progresses. (Item 76) The method according to any one of items 43 to 75, wherein the imaging includes imaging the amplicons embedded in the one or more hydrogels using confocal microscopy, two-photon microscopy, bright-field microscopy, intact tissue expansion microscopy, and / or CLARITY® optimized light-sheet microscopy (COLM). (Item 77) The method according to any one of items 43 to 76, wherein the intact tissue is a thin section. (Item 78) The method according to item 77, wherein the intact tissue has a thickness of 5 to 20 μm. (Item 79) The method according to item 77 or 78, wherein the contact of the amplicons embedded in one or more hydrogels occurs four or more times. (Item 80) The method according to item 77 or 78, wherein the amplicons embedded in one or more hydrogels are brought into contact five or more times. (Item 81) The method according to any one of items 43 to 76, wherein the intact tissue is a thick section. (Item 82) The method according to item 81, wherein the intact tissue has a thickness of 50 to 200 μm. (Item 83) The method according to item 81 or 82, wherein the amplicons embedded in the one or more hydrogels are brought into contact six or more times. (Item 84) The method according to item 81 or 82, wherein the amplicons embedded in one or more hydrogels are brought into contact seven or more times. (Item 85) The method according to any one of items 43 to 84, wherein the detection includes performing flow cytometry, sequencing, probe binding and electrochemical detection, pH change, catalysis induced by enzymes bound to DNA tags, quantum entanglement, Raman spectroscopy, terahertz wave techniques, and / or scanning electron microscopy. (Item 86) The flow cytometry is either mass cytometry or fluorescence-activated flow cytometry. The method described in item 85. (Item 87) The method according to any one of items 43 to 86, wherein the detection involves performing microscopy, scanning mass spectrometry, or other imaging techniques. (Item 88) The method according to any one of items 43 to 87, wherein the detection includes determining a signal. (Item 89) The method according to item 88, wherein the signal is a fluorescent signal. (Item 90) It is a system, A device including an imaging chamber and a pump, A system comprising a processor unit configured to perform one of items 1 through 42. [Brief explanation of the drawing] 【0007】 [Figure 1A] A schematic diagram of a spatially restructured transcription amplicon readout mapping (STARmap) method, such as those described herein, including in-situ RNA sequencing for spatial transcriptomics in a 3D tissue environment, is depicted. [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 1D] Same as above. [Figure 1E] Same as above. [Figure 2A] Depict nucleic acid specific amplification (SNAIL) probes via intramolecular ligation for high-quality RNA imaging and mouse brain instantiation. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 2D] Same as above. [Figure 2E] Same as above. [Figure 2F] Same as above. [Figure 2G] Same as above. [Figure 2H] Same as above. [Figure 2I] Same as above. [Figure 3A] This paper depicts hydrogel histochemistry (HTC) for background reduction and amplicon immobilization in STARmaps. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above. [Figure 3E] Same as above. [Figure 3F] Same as above. [Figure 3G] Same as above. [Figure 3H] Same as above. [Figure 4A] This paper describes the dynamic annealing and ligation-based sequencing (SEDAL) component of STARmap, which is a low-background error-corrected in situ sequencing component. [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above. [Figure 4E] Same as above. [Figure 4F] Same as above. [Figure 4G] Same as above. [Figure 4H] Same as above. [Figure 4I] Same as above. [Figure 4J] Same as above. [Figure 4K] Same as above. [Figure 4L] Same as above. [Figure 5A] This specification describes the classification of cell types within the primary visual cortex using the method described herein. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 5D] Same as above. [Figure 5E] Same as above. [Figure 5F] Same as above. [Figure 5G] Same as above. [Figure 5H] Same as above. [Figure 5I] Same as above. [Figure 5J] Same as above. [Figure 5K] Same as above. [Figure 5L] Same as above. [Figure 5M] Same as above. [Figure 5N] Same as above. [Figure 6A]Describe the data processing pipeline for STARmap. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 7A] Describe gene expression information for STAR mapping of inhibitory and excitatory subclusters. [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 7D] Same as above. [Figure 8A] This describes the subclustering of non-neuronal cell types. [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 9A] This specification describes the lack of batch effects with reproducible cell clustering using the method described herein. [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 9D] Same as above. [Figure 9E] Same as above. [Figure 9F] Same as above. [Figure 9G] Same as above. [Figure 10A] This document describes behavioral experiences in which cell type-specific regulation of regulated genes (ARGs) is detected and quantified using the methods described herein. [Figure 10B] Same as above. [Figure 10C] Same as above. [Figure 10D] Same as above. [Figure 11A] The expression information of dark samples and ARGs, as well as light samples and ARGs, is described using the methods described herein. [Figure 11B] Same as above. [Figure 11C] Same as above. [Figure 12] Describe the STARmap experimental flowchart for thin and thick tissue sections. [Figure 13A]This depicts a three-dimensional construct of cell types within the visual cortex volume. [Figure 13B] Same as above. [Figure 13C] Same as above. [Figure 13D] Same as above. [Figure 13E] Same as above. [Figure 13F] Same as above. [Figure 13G] Same as above. [Figure 13H] Same as above. [Figure 13I] Same as above. [Figure 14A] This depicts sequential SEDAL readout, which is the expression of multiple marker genes. [Figure 14B] Same as above. [Figure 15A] This paper describes 2D nearest neighbor analysis and cross-method validation of short-range inhibitory clusters in the mouse primary visual cortex. [Figure 15B] Same as above. [Figure 15C] Same as above. [Figure 16A] This describes the scalability of STARmap. [Figure 16B] Same as above. [Figure 16C] Same as above. [Figure 16D] Same as above. [Figure 16E] Same as above. [Figure 17] This describes the correlation between neuronal types identified by STARmap and publicly available single-cell RNA sequencing results. [Figure 18A] This document describes gene expression analysis of cell type subclusters in the medial prefrontal cortex (mPFC) using the methods described herein. [Figure 18B] Same as above. [Figure 18C] Same as above. [Figure 18D] Same as above. [Figure 18E] Same as above. [Figure 18F] Same as above. [Figure 19A]The reproducibility and spatial organization of cell clusters in mPFCs are described using the methods described herein. [Figure 19B] Same as above. [Figure 19C] Same as above. [Figure 20A] This specification describes the analysis of 1020 genes in a mouse hippocampal cell culture in 6 rounds of sequencing using the method described herein. [Figure 20B] Same as above. [Figure 20C] Same as above. [Figure 21A-1] This document describes gene expression analysis of 1020 genes in the mouse primary visual cortex using the methods described herein. [Figure 21A-2] Same as above. [Figure 21B-1] Same as above. [Figure 21B-2] Same as above. [Figure 21C-1] Same as above. [Figure 21C-2] Same as above. [Figure 21C-3] Same as above. [Figure 22A] This specification describes the reproducibility and cross-method comparison of measurements of 1020 genes in the mouse primary visual cortex using the methods described herein. [Figure 22B] Same as above. [Figure 22C] Same as above. [Figure 22D] Same as above. [Figure 23A] This document describes the STARmap experimental flowchart and loss integration for thin and thick tissue sections. [Figure 23B] Same as above. [Figure 23C] Same as above. [Figure 23D] Same as above. [Figure 24A] This section depicts a graphic layout of an exemplary fluid system used to carry out the methods described herein. [Figure 24B] Same as above. [Modes for carrying out the invention] 【0008】 Devices, methods, and systems for in-situ gene sequencing of target nucleic acids in cells within intact tissue are provided herein. Methods for screening candidate drugs to determine whether a candidate drug modulates gene expression of nucleic acids in cells within intact tissue are also provided herein. 【0009】 In some embodiments, a disclosed method for 3D intact tissue RNA sequencing in the brain and other organs, referred to as spatially degraded transcription amplicon readout mapping (STARmap), involves integrating a sequencing process with improved ligation, specific signal amplification, and hydrogel histochemistry (Figure 1A). In certain embodiments, STARmap enables cell-resolution expression mapping through sequencing of 160 different genes in all cells within a mouse visual cortex section. In certain embodiments, STARmap enables the identification of diverse anatomically and molecularly degraded cell types within the cortical layer, including interneuronal and glial subtypes, as well as the quantification of activity-regulated gene expression as a function of visual stimuli, spatial location, and molecularly defined cell type. In certain embodiments, STARmap enables the quantification of more than 30,000 cells in a cubic millimeter-scale volume, revealing 3D clustering patterns of inhibitory neurons and contrasting gradient distributions of excitatory neuron subtypes. 【0010】 In some embodiments, in situ-synthesized hydrogels are integrated with intracellularly delivered interfaces that bind to native biomolecules, thereby transforming the tissue from within its constituent cells into a new state suitable for high-resolution volumetric imaging and analysis, which is compliant with many types of molecular phenotypic analysis for proteins, nucleic acids, and other targets. For example, synthetic hydrogels have been used to accommodate enzymatic reactions, including DNA sequencing, and are known in the art, including, but are not limited to, the techniques disclosed in WO2014 / 025392, the reference of which is incorporated herein by reference. In some embodiments, biological tissues can be transformed into forms embedded in hydrogels, which are compliant with the creation, retention, and functional presentation of RNA-derived complementary DNA (cDNA). In such embodiments, 3D in situ sequencing can be performed within such tissue-hydrogel formulations, thus taking advantage of important associated properties such as optical clarity, reduced background, increased diffusion rate, and greater mechanical stability. 【0011】 The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein and refer to polymeric forms of amino acids of any length, which may include encoded and unencoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a modified peptide backbone. The term “polypeptide” includes, but is not limited to, fusion proteins, including fusion proteins having heterologous amino acid sequences, fusions with heterologous and homologous leader sequences with or without an N-terminal methionine residue, and immunologically tagged proteins. The term “polypeptide” includes polypeptides comprising one or more fatty acid moieties, lipid moieties, sugar moieties, and carbohydrate moieties. The term “polypeptide” includes post-translationally modified polypeptides. 【0012】 As used herein, the term “target nucleic acid” refers to any polynucleotide nucleic acid molecule present in a single cell (e.g., DNA molecule, RNA molecule, modified nucleic acid, etc.). In some embodiments, the target nucleic acid is coding RNA (e.g., mRNA). In some embodiments, the target nucleic acid is non-coding RNA (e.g., tRNA, rRNA, microRNA (miRNA), mature miRNA, immature miRNA, etc.). In some embodiments, the target nucleic acid is a splice mutation of an RNA molecule (e.g., mRNA, premRNA, etc.) in the context of a cell. Thus, suitable target nucleic acids may be unspliced ​​RNA (e.g., premRNA, mRNA), partially spliced ​​RNA, or fully spliced ​​RNA, etc. The target nucleic acid of interest can be expressed variably within a cell population, i.e., may have different abundances, where the method of the present invention enables profiling and comparison of nucleic acid expression levels, including but not limited to RNA transcripts, in individual cells. The target nucleic acid may also be a DNA molecule, e.g., a mutated genome, a virus, a plasmid, etc. For example, these methods can be used to detect virus-infected cells, for instance, to determine copy number variations, viral load, and dynamics in cancer cell populations where target nucleic acids are present in varying amounts in the genomes of cells within the population. 【0013】 As used interchangeably herein, the terms “oligonucleotide,” “polynucleotide,” and “nucleic acid molecule” refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Therefore, these terms include, but are not limited to, polymers containing single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or purine and pyrimidine bases, or other natural, chemically, or biochemically modified, unnatural, or derivatized nucleotide bases. The polynucleotide backbone may contain sugars and phosphate groups (typically found in RNA or DNA), or modified or substituted sugars or phosphate groups. Alternatively, the polynucleotide backbone may contain polymers of synthetic subunits such as phosphoramidites and / or phosphorothioates, and thus may be oligodeoxynucleoside phosphoramidates or mixed phosphoramidate phosphodiester oligomers. Peyrottes et al. (1996) Nucl. Acids Res. 24:1841-1848, Chaturvedi et al. (1996) Nucl. Acids Res.24:2318-2323. Polynucleotides may contain one or more L-nucleosides. Polynucleotides may contain modified nucleotides such as methylated nucleotides and nucleotide analogs, uracil, other sugars, and linking groups such as fluororibose and thioates, as well as nucleotide branching. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be modified to contain N3'-P5'(NP) phosphoramidates, morpholinophosphorosidates (MF), roch nucleic acids (LNA), 2'-O-methoxyethyl (MOE), or 2'-fluoroarabino nucleic acids (FANA), which can enhance the resistance of polynucleotides to nuclease degradation (see, e.g., Faria et al. (2001) Nature Biotechnol. 19:40-44, Toulme (2001) Nature Biotechnol. 19:17-18). Polynucleotides can be further modified after polymerization, such as by conjugation with labeling components. Other types of modifications included in this definition are capping, substitution of one or more naturally occurring nucleotides with analogs, and introduction of means for binding the polynucleotide to a protein, metal ion, labeling component, other polynucleotide, or solid support. Immunomodulatory nucleic acid molecules can be provided in various formulations, such as microencapsulated in association with liposomes, as described in more detail herein. The polynucleotides used for amplification are generally single-stranded for maximum efficiency in amplification, but may alternatively be double-stranded. If double-stranded, the polynucleotide may be treated to first separate its strands before being used to prepare the extension product. This denaturation step is typically performed with heat, but can alternatively be carried out by using an alkali followed by neutralization. 【0014】 "Subject," "individual," or "patient" refers to any subject for which treatment is desired. Of particular interest are human subjects. Other subjects may include non-human primates, cattle, sheep, goats, dogs, cats, birds (e.g., chickens or other poultry), guinea pigs, rabbits, rats, mice, and horses. Of particular interest are subjects with or susceptible to brain damage. 【0015】 Before further description of the present invention, it should be understood that the present invention is not limited to the specific embodiments described and is therefore naturally subject to change. It should also be understood that the terms used herein are for the purpose of describing only specific embodiments and are not intended to limit the scope of the present invention, as it is limited only by the appended claims. 【0016】 Where a range of values ​​is provided, unless the context explicitly indicates otherwise, it is understood that the upper and lower limits of that range, and any other described values ​​or intervening values ​​within that described range, up to one-tenth of the lower limit, are included in the invention. These smaller upper and lower limits may be independently included in smaller ranges, depending on any specifically excluded limits within the described range, and are also included in the invention. Where a described range includes one or both of the limits, the range excluding one or both of those included limits is also included in the invention. 【0017】 Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Any methods and materials similar or equivalent to those described herein may also be used in the performance or testing of the present invention, but preferred methods and materials are described herein. All publications referenced herein are incorporated herein by reference to disclose and illustrate methods and / or materials in relation to the cited publications. 【0018】 It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural nouns unless otherwise explicitly stated in the context. Thus, for example, a reference to “cells” includes multiple such cells, and a reference to “the oligonucleotides” includes one or more oligonucleotides and their equivalents known to those skilled in the art. It should be further noted that claims may be constructed to exclude any optional elements. Therefore, this statement is intended to serve as an antecedent for the use of such exclusive terms as “simply,” “only,” or “negative” limitation relating to the enumeration of claim elements. 【0019】 For clarity, certain features of the Invention described in the context of separate embodiments may be provided in combination in a single embodiment. Conversely, various features of the Invention described in the context of a single embodiment for brevity may be provided separately or in any preferred partial combination. All combinations of embodiments relating to the Invention are specifically encompassed by the Invention and are disclosed herein as if every conceivable combination were individually and expressly disclosed. In addition, various embodiments and all partial combinations of their elements are also specifically encompassed by the Invention and are disclosed herein as if every conceivable such partial combination were individually and expressly disclosed herein. 【0020】 The publications discussed herein are provided only for disclosures prior to the filing date of this application. Nothing herein should be construed as acknowledging that the present invention has no prior rights to such publications for the sake of prior art. Furthermore, the dates of the publications provided may differ from the actual publication dates, and these may need to be verified independently. 【0021】 method Methods disclosed herein include image-based in situ nucleic acid (DNA and / or RNA) sequencing techniques, including an improved ligation-based sequencing process, specific signal amplification, hydrogel histochemistry for transforming biological tissues into transparent sequencing chips, and an associated data analysis pipeline for spatially resolving highly multiplexed gene detection at the subcellular and cellular levels, referred to as "Spatial Resolved Transcription Amplicon Readout Mapping (STARmap)." In some embodiments, STARmap defines a platform for 3D in situ transcriptomics, enabled by improved cDNA library preparation, sequencing, and hydrogel histochemistry. 【0022】 As summarized above, the method disclosed herein includes a method for in situ gene sequencing of a target nucleic acid in cells within intact tissue, the method comprising (a) contacting immobilized and permeabilized intact tissue with at least a pair of oligonucleotide primers under conditions that enable specific hybridization, wherein the primer pair comprises a first oligonucleotide and a second oligonucleotide, each of which comprises a first complementary region, a second complementary region, and a third phase The first oligonucleotide comprises a complementation region, the second oligonucleotide further comprises a barcode sequence, the first complementation region of the first oligonucleotide is complementary to the first portion of the target nucleic acid, the second complementation region of the first oligonucleotide is complementary to the first complementation region of the second oligonucleotide, the third complementation region of the first oligonucleotide is complementary to the third complementation region of the second oligonucleotide, the second complementation region of the second oligonucleotide is complementary to the second portion of the target nucleic acid, and the first complementation region of the first oligonucleotide is... (b) contacting the second complementary region of the second oligonucleotide adjacent to it, (c) adding a ligase to ligate the second oligonucleotide and generate a closed nucleic acid ring, (d) performing rolling circle amplification in the presence of nucleic acid molecules, using the second oligonucleotide as a template and the first oligonucleotide as a primer for polymerase to form one or more amplicons, (e) embedding one or more amplicons in the presence of a hydrogel subunit to form amplicons embedded in one or more hydrogels, (e) contacting the amplicons embedded in one or more hydrogels having a barcode sequence with a pair of primers under conditions that allow ligation, the pair of primers comprising a third oligonucleotide and a fourth oligonucleotide, and ligation occurs only when both the third oligonucleotide and the fourth oligonucleotide ligate to the same amplicon, and (f) repeating step (e) many times.(g) The process includes imaging amplicons embedded in one or more hydrogels to determine the in-situ gene sequence of target nucleic acids in cells within intact tissue. 【0023】 The method disclosed herein also provides a method for screening candidate drugs to determine whether a candidate drug modulates gene expression of nucleic acids in cells within intact tissue, the method comprising (a) contacting immobilized and permeabilized intact tissue with at least a pair of oligonucleotide primers under conditions that enable specific hybridization, wherein the primer pair comprises a first oligonucleotide and a second oligonucleotide, each of the first and second oligonucleotides comprising a first complementary region and a second complementary region The second oligonucleotide further comprises a barcode sequence, the first complementary region of the first oligonucleotide being complementary to the first portion of the target nucleic acid, the second complementary region of the first oligonucleotide being complementary to the first complementary region of the second oligonucleotide, the third complementary region of the first oligonucleotide being complementary to the third complementary region of the second oligonucleotide, the second complementary region of the second oligonucleotide being complementary to the second portion of the target nucleic acid, and the first complementary region of the first oligonucleotide (b) Contacting the second oligonucleotide having a sexual region adjacent to the second complementary region of the second oligonucleotide; (c) Adding a ligase to ligate the second oligonucleotide and generate a closed nucleic acid ring; (d) Performing rolling circle amplification in the presence of nucleic acid molecules, using the second oligonucleotide as a template and the first oligonucleotide as a primer for polymerase to form one or more amplicons; (e) Embedding one or more amplicons in the presence of a hydrogel subunit to form amplicons embedded in one or more hydrogels; (e) Contacting the amplicons embedded in one or more hydrogels having a barcode sequence with a pair of primers under conditions that allow ligation, the pair of primers comprising a third oligonucleotide and a fourth oligonucleotide, and ligation occurs only when both the third oligonucleotide and the fourth oligonucleotide ligate to the same amplicon; and (f) Repeating step (e) many times.(g) imaging amplicons embedded in one or more hydrogels to determine the in-situ gene sequence of a target nucleic acid in cells within intact tissue; and (h) detecting the level of gene expression of the target nucleic acid, wherein the change in the level of expression of the target nucleic acid in the presence of at least one candidate drug compared to the level of expression of the target nucleic acid in the absence of at least one candidate drug indicates that at least one candidate drug modulates gene expression of the nucleic acid in cells within intact tissue. 【0024】 In certain embodiments, the methods disclosed herein offer faster processing times, higher multiplicity (up to 1000 genes), higher efficiency, higher sensitivity, lower error rates, and more spatially resolved cell types compared to existing gene expression analysis tools. In such embodiments, the improved hydrogel histochemistry method is a sequencing process by improved ligation (SEDAL) for in-situ sequencing with reduced error, transforming biological tissues with nucleic acids imprinted on hydrogels suitable for in-situ sequencing. In several other embodiments, the methods disclosed herein include spatial sequencing (e.g., reagents, chips, or services) for biomedical research and clinical diagnosis (e.g., cancer, bacterial infections, viral infections, etc.) with single-cell and / or single-molecule sensitivity. 【0025】 Nucleic acid specific amplification via intramolecular ligation (SNAIL) In some embodiments, one component of STARmap may be referred to as SNAIL, which stands for Specific Amplification of Nucleic Acids via Intramolecular Ligation, and includes an efficient approach for generating a cDNA library in situ from cellular RNA. In certain embodiments, the method of the present invention includes contacting an immobilized and permeabilized intact tissue with at least a pair of oligonucleotide primers under conditions that allow for specific hybridization, wherein the pair of primers comprises a first oligonucleotide and a second oligonucleotide. 【0026】 More generally, nucleic acids present in target cells within a tissue serve as a scaffold for assembling a complex comprising a pair of primers, referred herein as a first oligonucleotide and a second oligonucleotide. In some embodiments, contacting immobilized and permeabilized intact tissue involves hybridizing the pair of primers to the same target nucleic acid. In some embodiments, the target nucleic acid is RNA. In such embodiments, the target nucleic acid may be mRNA. In other embodiments, the target nucleic acid is DNA. 【0027】 As used herein, the terms “hybridize” and “hybridization” refer to the formation of a complex between nucleotide sequences that are sufficiently complementary to form a complex via Watson-Crick base pairing. When a primer “hybridizes” with its target (template), such a complex (or hybrid) is sufficiently stable to perform the priming function required by DNA polymerase, for example, to initiate DNA synthesis. It will be understood that the hybridization sequences do not need to have perfect complementarity to provide a stable hybrid. In many situations, a stable hybrid will be formed when less than about 10% of the bases are mismatched and loops of four or more nucleotides will be ignored. Thus, as used herein, the term “complementary” refers to oligonucleotides that form a stable double helix in “complementary” form under assay conditions, generally when there is about 90% or more homology. 【0028】 SNAIL oligonucleotide primer In the method of the present invention, the SNAIL oligonucleotide primer comprises at least a first oligonucleotide and a second oligonucleotide, each of which comprises a first complementary region, a second complementary region, and a third complementary region, the second oligonucleotide further comprising a barcode sequence, the first complementary region of the first oligonucleotide being complementary to a first portion of the target nucleic acid, the second complementary region of the first oligonucleotide being complementary to the first complementary region of the second oligonucleotide, the third complementary region of the first oligonucleotide being complementary to the third complementary region of the second oligonucleotide, the second complementary region of the second oligonucleotide being complementary to a second portion of the target nucleic acid, and the first complementary region of the first oligonucleotide being adjacent to the second complementary region of the second oligonucleotide. In an alternative embodiment, the second oligonucleotide is a closed cyclic molecule, and the ligation step is omitted. 【0029】 This disclosure provides a method comprising contacting an immobilized and permeabilized tissue to hybridize a plurality of oligonucleotide primers having specificity for different target nucleic acids. In some embodiments, the method includes a plurality of first oligonucleotides, including, but not limited to, five or more first oligonucleotides that hybridize to target nucleotide sequences, e.g., eight or more, ten or more, twelve or more, fifteen or more, eighteen or more, twenty or more, twenty or more, thirty or more, thirty or more, thirty or more, thirty or more. In some embodiments, the method of this disclosure includes a plurality of first oligonucleotides, including, but not limited to, fifteen or more first oligonucleotides that hybridize to 15 or more, e.g., 20 or more, thirty or more, forty or more, fifty or more, sixty or more, seventy or more, and up to eighty different target nucleic acid sequences, e.g., 20 or more, thirty or more, forty or more, fifty or more, sixty or more, seventy or more, and up to eighty different first oligonucleotides. In some embodiments, the method includes, but is not limited to, a plurality of second oligonucleotides comprising five or more second oligonucleotides, e.g., eight or more, ten or more, twelve or more, fifteen or more, eighteen or more, twenty or more, twenty-five or more, thirty or more, thirty or more, or thirty or more. In some embodiments, the method of the present disclosure includes, but is not limited to, a plurality of second oligonucleotides comprising 15 or more second oligonucleotides, e.g., twenty or more, thirty or more, forty or more, fifty or more, sixty or more, seventy or more, and up to eighty different first oligonucleotides that hybridize to fifteen or more, e.g., twenty or more, thirty or more, forty or more, fifty or more, sixty or more, seventy or more, and up to eighty different target nucleic acid sequences. If one or more pairs specifically bind to each target nucleic acid, a plurality of oligonucleotide pairs can be used in the reaction. For example, two primer pairs can be used for one target nucleic acid to improve sensitivity and reduce variability.It is also an object to detect a plurality of different target nucleic acids in a cell, for example, to detect up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 12, up to 15, up to 18, up to 20, up to 25, up to 30, up to 40 or more different target nucleic acids. Primers are typically denatured by heating to a temperature of at least about 50°C, at least about 60°C, at least about 70°C, at least about 80°C, and up to about 99°C, up to about 95°C, up to about 90°C prior to use. 【0030】 In some embodiments, the primer is denatured by heating prior to contacting with the sample. In certain embodiments, the melting temperature (T m ) of the oligonucleotide is selected to minimize ligation in solution. The "melting temperature" or "Tm" of a nucleic acid is defined as the temperature at which half of the helical structure of the nucleic acid is lost due to heating or other dissociation of the hydrogen bonds between base pairs, such as by treatment with an acid or an alkali. The T m of a nucleic acid molecule depends on its length and its base composition. A nucleic acid molecule rich in GC base pairs has a higher T m than one with a rich AT base pair. When the temperature is below T m , the separated complementary strands of the nucleic acid spontaneously reassociate or anneal to form a double-stranded nucleic acid. The highest rate of nucleic acid hybridization occurs at a temperature about 25°C lower than T m . T m can be estimated using the following relationship. T m = 69.3 + 0.41(GC)% (Marmur et al. (1962) J. Mol. Biol. 5: 109 - 118). 【0031】 In certain embodiments, a plurality of second oligonucleotides comprise a padlock probe. In some embodiments, the probe comprises a detectable label that can be measured and quantified. The terms “label” and “detectable label” refer to detectable molecules, including, but not limited to, radioisotopes, fluorescent agents, chemiluminescent agents, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal solvents, ligands (e.g., biotin or hapten). The term “fluorescent agent” refers to a substance or a portion thereof that is capable of exhibiting 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 dye, fluorescein, YPet, CyPet, Cascade Blue, allophycocyanin, Cy3, Cy5, Cy7, rhodamine, dansyl, umbelliferone, Texas Red, luminol, acradium ester, biotin, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), firefly cyferase, renyral cyferase, NADPH, beta-galactosidase, horadish peroxidase, glucose oxidase, alkaline phosphatase, chloramphene acetyltransferase, and urelases. 【0032】 In some embodiments, one or more first and second oligonucleotides bind to different regions or target sites of the target nucleic acid. In a pair, each target site is different and is an adjacent site on the target nucleic acid, typically located no more than 15 nucleotides away from the other site, e.g., 10, 8, 6, 4, or 2 nucleotides away. The target sites are typically located on the same strand of the target nucleic acid in the same direction. The target sites are also selected to provide unique binding sites compared to other nucleic acids present in the cell. Each target site is generally about 19 to 25 nucleotides long, e.g., about 19 to 23 nucleotides, about 19 to 21 nucleotides, or about 19 to 20 nucleotides. The pair of first and second oligonucleotides is selected such that each oligonucleotide in the pair has a similar melting temperature for binding to its congeneral target site, e.g., T m The temperature may be approximately 50°C, 52°C, 58°C, 62°C, 65°C, 70°C, or 72°C. The GC content of the target site is generally selected to be approximately 20% or less, 30% or less, 40% or less, 50% or less, 60% or less, or 70% or less. 【0033】 In some embodiments, the first oligonucleotide includes first, second, and third complementary regions. The target site of the first oligonucleotide can point to the first complementary region. As summarized above, the first complementary region of the first oligonucleotide may have a length of 19 to 25 nucleotides. In certain embodiments, the second complementary region of the first oligonucleotide has a length of 3 to 10 nucleotides, for example, containing 4 to 8 nucleotides or 4 to 7 nucleotides. In some embodiments, the second complementary region of the first oligonucleotide has a length of 6 nucleotides. In some embodiments, the third complementary region of the first oligonucleotide similarly has a length of 6 nucleotides. In such embodiments, the third complementary region of the first oligonucleotide has a length of 3 to 10 nucleotides, for example, containing 4 to 8 nucleotides or 4 to 7 nucleotides. 【0034】 In some embodiments, the second oligonucleotide comprises first, second, and third complementary regions. The target site of the second oligonucleotide can point to the second complementary region. As summarized above, the second complementary region of the second oligonucleotide may have a length of 19 to 25 nucleotides. In certain embodiments, the first complementary region of the first oligonucleotide has a length of 3 to 10 nucleotides, for example, containing 4 to 8 nucleotides or 4 to 7 nucleotides. In some embodiments, the first complementary region of the first oligonucleotide has a length of 6 nucleotides. In some embodiments, the first complementary region of the second oligonucleotide comprises the 5' end of the second oligonucleotide. In some embodiments, the third complementary region of the second oligonucleotide similarly has a length of 6 nucleotides. In such embodiments, the third complementary region of the second oligonucleotide has a length of 3 to 10 nucleotides, for example, containing 4 to 8 nucleotides or 4 to 7 nucleotides. In further embodiments, the third complementary region of the second oligonucleotide includes the 3' end of the second oligonucleotide. In some embodiments, the first complementary region of the second oligonucleotide is adjacent to the third complementary region of the second oligonucleotide. 【0035】 In some embodiments, the second oligonucleotide comprises a barcode sequence, the barcode sequence of the second oligonucleotide providing barcoded information for the identification of a 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 ligated to the target nucleic acid of interest during amplification and used to trace the amplicon to the cell from which the target nucleic acid originates. The barcode sequence can be added to the target nucleic acid of interest during amplification by performing amplification with an oligonucleotide containing a region with the barcode sequence and a region complementary to the target nucleic acid, so that the barcode sequence is incorporated into the final amplified target nucleic acid product (i.e., the amplicon). 【0036】 organization As described herein, the disclosed method includes an in-situ sequencing technique for intact tissue by contacting the fixed and permeabilized intact tissue with at least one pair of oligonucleotide primers under conditions that allow for specific hybridization. Suitable tissue specimens for use in the method described herein generally include, but are not limited to, any type of tissue specimen collected from living or cadavers, such as biopsy specimens and autopsy specimens, including epithelial, muscle, connective tissue, and nerve tissue. The tissue specimen may be collected and processed using the method described herein and subjected to microscopic analysis immediately after processing, or it may be stored and subjected to microscopic analysis at a future point in time, for example, after long-term storage. In some embodiments, the method described herein may be used to preserve the tissue specimen in a stable, accessible, and completely intact form for future analysis. In some embodiments, the method described herein may be used to analyze previously preserved or stored tissue specimens. In some embodiments, the intact tissue includes brain tissue, such as visual cortical sections. In some embodiments, intact tissue is a thin section 5–20 μm thick, including, but not limited to, 5–18 μm, 5–15 μm, or 5–10 μm. In other embodiments, intact tissue is a thick section 50–200 μm thick, including, but not limited to, 50–150 μm, 50–100 μm, or 50–80 μm. 【0037】 Aspects of the present invention include fixing intact tissue. As used herein, the term “fixing” or “immobilization” refers to the process of protecting biological material (e.g., tissues, cells, organs, molecules, etc.) from decay and / or degradation. Fixation can be achieved using any convenient protocol. Fixation may include contacting a sample with a fixation reagent (i.e., a reagent containing at least one fixative). A sample may be in contact with the fixation reagent for a wide range of times, which may depend on temperature, the properties of the sample, and the fixative(s). For example, a sample may be in contact with the fixation reagent for 24 hours or less, 18 hours or less, 12 hours or less, 8 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, 60 hours or less, 45 hours or less, 30 hours or less, 25 hours or less, 20 hours or less, 15 hours or less, 10 hours or less, 5 hours or less, or 2 hours or less. 【0038】 The sample can be in contact with the fixative for a period of time ranging from 5 minutes to 24 hours, for example, 10 minutes to 20 hours, 10 minutes to 18 hours, 10 minutes to 12 hours, 10 minutes to 8 hours, 10 minutes to 6 hours, 10 minutes to 4 hours, 10 minutes to 2 hours, 15 minutes to 20 hours, 15 minutes to 18 hours, 15 minutes to 12 hours, 15 minutes to 8 hours, 15 minutes to 6 hours, 15 minutes to 4 hours, 15 minutes to 2 hours, 15 minutes to 1 hour, 15 minutes to 1.5 hours, 15 minutes to 1 hour, 10 minutes to 30 minutes, 15 minutes to 30 minutes, 30 minutes to 2 hours, 45 minutes to 1.5 hours, or 55 minutes to 70 minutes. 【0039】 The sample can be contacted with the fixative at various temperatures, depending on the protocol and reagents used. For example, in some cases, the sample can be contacted with the fixative at temperatures in the range of -22°C to 55°C, and the specific range of interest may include, but is not limited to, 50–54°C, 40–44°C, 35–39°C, 28–32°C, 20–26°C, 0–6°C, and -18–22°C. In some cases, the sample can be contacted with the fixative at temperatures of -20°C, 4°C, room temperature (22–25°C), 30°C, 37°C, 42°C, or 52°C. 【0040】 Any convenient fixation reagent can be used. Common fixation reagents include crosslinking agents, precipitation agents, oxidative agents, and mercury. Crosslinking agents chemically bond two or more molecules by covalent bonds, and a wide range of crosslinking reagents can be used. Examples of suitable crosslinking agents, but not limited to these, include aldehydes (e.g., formaldehyde, glutaraldehyde, etc., also commonly referred to as "paraformaldehyde" and "formalin"), imide esters, and NHS (N-hydroxysuccinimide) esters. Examples of suitable precipitation agents, but not limited to these, include alcohols (e.g., methanol, ethanol), acetone, and acetic acid. In some embodiments, the fixation agent is formaldehyde (i.e., paraformaldehyde or formalin). Preferred final concentrations of formaldehyde in the fixation reagent are 0.1–10%, 1–8%, 1–4%, 1–2%, 3–5%, or 3.5–4.5%, containing approximately 1.6% after 10 minutes. In some embodiments, the sample is fixed with a final concentration of 4% formaldehyde (e.g., when diluted from a more concentrated stock solution, such as 38%, 37%, 36%, 20%, 18%, 16%, 14%, 10%, 8%, 6%). In some embodiments, the sample is fixed with a final concentration of 10% formaldehyde. In some embodiments, the sample is fixed with a final concentration of 1% formaldehyde. In some embodiments, the fixative is glutaraldehyde. A preferred concentration of glutaraldehyde in the fixative reagent is 0.1–1%. The fixative reagent may contain two or more fixatives in any combination. For example, in some embodiments, the sample is brought into contact with a fixative reagent containing both formaldehyde and glutaraldehyde. 【0041】 As used herein, the terms “permeabilization” or “permeabilization treatment” refer to the process of rendering cells (such as cell membranes) of a sample permeable to experimental reagents such as nucleic acid probes, antibodies, and chemical substrates. Any convenient method and / or reagent can be used for permeabilization. Suitable permeabilization reagents include detergents (e.g., saponins, Triton X-100, Tween-20, etc.), organic fixatives (e.g., acetone, methanol, ethanol, etc.), and enzymes. Detergents can be used in a range of concentrations. For example, 0.001% to 1%, 0.05% to 0.5%, or 0.1% to 0.3% detergents can be used for permeabilization (e.g., 0.1% saponin, 0.2% Tween-20, 0.1% to 0.3% Triton X-100, etc.). In some embodiments, permeabilization is performed using methanol on ice for at least 10 minutes. 【0042】 In some embodiments, the same solution can be used as both a fixation reagent and a permeabilization reagent. For example, in some embodiments, the fixation reagent contains 0.1% to 10% formaldehyde and 0.001% to 1% saponin. In some embodiments, the fixation reagent contains 1% formaldehyde and 0.3% saponin. 【0043】 The sample can be in contact with the permeabilizing reagent for a wide range of times, which may depend on the temperature, the properties of the sample, and the permeabilizing reagent(s). For example, the sample can be in contact with the permeabilizing reagent for 24 hours or more, 24 hours or less, 18 hours or less, 12 hours or less, 8 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, 60 minutes or less, 45 minutes or less, 30 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, or 2 minutes or less. The sample can be in contact with the permeabilizing reagent at various temperatures, depending on the protocol and reagents used. For example, in some cases, the sample can be in contact with the permeabilizing reagent at temperatures in the range of -82°C to 55°C, and the specific range of interest may include, 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, -18 to -22°C, and -78 to -82°C. In some cases, the sample can be contacted with a permeabilizing reagent at temperatures of -80°C, -20°C, 4°C, room temperature (22-25°C), 30°C, 37°C, 42°C, or 52°C. 【0044】 In some embodiments, the sample is contacted with an enzyme permeabilization reagent. The enzyme permeabilization reagent permeates the sample by partially degrading the extracellular matrix or surface proteins that would otherwise hinder the permeation of the sample by the assay reagent. Contact with the enzyme permeabilization reagent can be performed at any point after fixation and before target detection. In some cases, the enzyme permeabilization reagent is the commercially available enzyme proteinase K. In such cases, the sample is contacted with proteinase K after fixation and before contact with the reagent. Proteinase K treatment (i.e., contact with proteinase K, also commonly referred to as "proteinase K digestion") can be performed over a range of time, at a range of temperatures, and within a range of enzyme concentrations empirically determined for each cell or tissue type under investigation. For example, the sample may 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 concentrations 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 temperatures in the range of 2°C to 55°C, and specific 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 temperatures of 4°C, room temperature (22 to 25°C), 30°C, 37°C, 42°C, or 52°C. In some embodiments, the sample does not come into contact with the enzyme permeabilization reagent. In some embodiments, the sample does not come into contact with proteinase K. Contact between intact tissue and at least a fixation reagent and a permeabilization reagent results in the formation of fixed and permeabilized tissue. 【0045】 Ligauze In some embodiments, the disclosed method includes adding a ligase to ligate a second oligonucleotide to generate a closed nucleic acid ring. In some embodiments, adding a ligase includes adding a DNA ligase. In alternative embodiments, the second oligonucleotide is provided as a closed nucleic acid ring, and the step of adding a ligase is omitted. In certain embodiments, the ligase is an enzyme that facilitates sequencing of the target nucleic acid molecule. 【0046】 As used herein, the term “ligase” refers to an enzyme commonly used to join polynucleotides together or to join the ends of a single polynucleotide. Examples of ligases include ATP-dependent double-stranded polynucleotide ligases, NAD-i-dependent double-stranded DNA or RNA ligases, and single-stranded polynucleotide ligases, e.g., any of the ligases described in EC6.5.1.1 (ATP-dependent ligases), EC6.5.1.2 (NAD+-dependent ligases), and EC6.5.1.3 (RNA ligases). Specific examples of ligases include bacterial ligases, e.g., E. coli DNA ligase and TaqDNA ligase, Ampligase® heat-stable DNA ligase (Epicentre® Technologies Corp., part of Illuminah®, Madison, Wisconsin), and phage ligases, e.g., T3DNA ligase, T4DNA ligase, and T7DNA ligase, and their variants. 【0047】 Rolling Circle Amplification In some embodiments, the method of the present invention comprises the step of performing rolling circle amplification in the presence of a nucleic acid molecule, which includes using a second oligonucleotide as a template and a first oligonucleotide as a primer so that the polymerase forms one or more amplicons. In such embodiments, a single-stranded cyclic polynucleotide template is formed by ligation of a second nucleotide, and this cyclic polynucleotide includes a region complementary to the first oligonucleotide. Upon addition of DNA polymerase in the presence of a suitable dNTP precursor and other cofactors, the first oligonucleotide is elongated by replication of numerous copies of the template. This amplification product can be readily detected by binding to a detection probe. 【0048】 In some embodiments, the second oligonucleotide can be cyclized and rolled-circle amplified only if the first and second oligonucleotides hybridize to the same target nucleic acid molecule, thereby generating cDNA nanoballs (i.e., amplicons) containing multiple copies of cDNA. The term "amplicon" refers to the amplified nucleic acid product of a PCR reaction or other nucleic acid amplification process. In some embodiments, amine-modified nucleotides are spiked into the rolling-circle amplification reaction. 【0049】 Techniques for rolling circle amplification are known in the art (e.g., Baner et al, Nucleic Acids Research, 26:5073-5078, 1998; Lizardi et al, Nature Genetics 19:226, 1998; Schweitzer et al. Proc. Natl See Acad.Sci.USA 97:10113-119,2000;Faruqi et al, BMC Genomics 2:4,2000;Nallur et al, Nucl.Acids Res.29:el18,2001;Dean et al.Genome Res.11:1095-1099,2001;Schweitzer et al, Nature Biotech .20:359-365,2002;U.S. Patents 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. 【0050】 In certain embodiments, the nucleic acid molecule comprises an amine-modified nucleotide. In such embodiments, the amine-modified nucleotide comprises a partial modification of N-hydroxysuccinimide acrylate. 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. 【0051】 Amplicon Embedding into Tissue-Hydrogel Setting In some embodiments, the disclosed method involves embedding one or more amplicons in the presence of hydrogel subunits to form amplicons embedded in one or more hydrogels. The described hydrogel histochemistry involves covalently bonding nucleic acids to in-situ synthesized hydrogels for tissue clarification, enzymatic diffusion, and multicycle sequencing, whereas existing hydrogel histochemistry methods are unable to do so. In some embodiments, to enable amplicon embedding into tissue-hydrogel settings, amine-modified nucleotides are spiked into a rolling-circle amplification reaction, functionalized with an acrylamide moiety using N-hydroxysuccinimide acrylate, copolymerized with an acrylamide monomer to form a hydrogel. 【0052】 As used herein, the terms “hydrogel” or “hydrogel network” mean a network of water-soluble polymer chains, sometimes seen as colloidal gels with water as the dispersion medium. In other words, hydrogels are a class of polymer materials that can absorb large amounts of water without dissolving. Hydrogels can contain more than 99% water and may contain natural or synthetic polymers, or combinations thereof. Hydrogels also possess a degree of flexibility very similar to that of natural tissues due to their significant water content. A detailed description of suitable hydrogels can be found in published U.S. Patent Application No. 2010 / 0055733, which is specifically incorporated herein by reference. As used herein, the terms “hydrogel subunit” or “hydrogel precursor” mean a hydrophilic monomer, prepolymer, or polymer that can be crosslinked or “polymerized” to form a three-dimensional (3D) hydrogel network. While not bound by any scientific theory, this fixation of a biological specimen in the presence of hydrogel subunits is thought to crosslink the components of the specimen to the hydrogel subunits, thereby fixing molecular components in place and preserving tissue structure and cellular morphology. 【0053】 In some embodiments, embedding involves copolymerizing one or more amplicons with acrylamide. As used herein, the term “copolymer” describes a polymer containing two or more types of subunits. This term encompasses polymers containing two, three, four, five, or six types of subunits. 【0054】 In certain embodiments, embedding includes clarifying an amplicon embedded in one or more hydrogels, wherein the target nucleic acid is substantially retained within the amplicon embedded in one or more hydrogels. In such embodiments, clarification includes substantially removing a plurality of cellular components from the amplicon embedded in one or more hydrogels. In some other embodiments, clarification includes substantially removing lipids from the amplicon embedded in one or more hydrogels. As used herein, the term “substantially” means that the original amount present in the sample before clarification has been reduced by about 70% or more, e.g., 75% or more, e.g., 80% or more, e.g., 85% or more, e.g., 90% or more, e.g., 95% or more, e.g., 99% or more, e.g., 100%. 【0055】 In some embodiments, clarifying an amplicon embedded in a hydrogel involves electrophoresis of the sample. In some embodiments, the amplicon is electrophoresed using a buffer containing an ionic surfactant. In some embodiments, the ionic surfactant is sodium dodecyl sulfate (SDS). In some embodiments, the sample is electrophoresed using a voltage in the range of about 10 to about 60 volts. In some embodiments, the sample is electrophoresed for a period in the range of about 15 minutes to about 10 days. In some embodiments, this method further includes incubating the clarified sample in a mounting medium having a refractive index that matches the refractive index of the clarified tissue. In some embodiments, the mounting medium increases the optical clarity of the sample. In some embodiments, the mounting medium contains glycerol. 【0056】 Sequence determination with error correction by dynamic annealing and ligation (SEDAL) A method disclosed herein comprises the step of contacting an amplicon embedded in one or more hydrogels having a barcode sequence with a pair of primers, under conditions that enable ligation, wherein the pair of primers comprises a third oligonucleotide and a fourth oligonucleotide, and ligation occurs only when both the third and fourth oligonucleotides ligate to the same amplicon. In some embodiments, SEDAL was specifically designed for STARmap. In such embodiments, the improved ligation sequencing method herein includes operating at room temperature for best preservation of tissue morphology with low background noise and error reduction. In other such embodiments, contacting the amplicons embedded in one or more hydrogels includes eliminating the accumulation of errors as sequencing progresses. 【0057】 In some embodiments, contact of amplicons embedded in one or more hydrogels occurs two or more times, including, but not limited to, three or more, four or more, five or more, six or more, or seven or more times. In certain embodiments, contact of amplicons embedded in one or more hydrogels occurs four or more times for thin tissue samples. In other embodiments, contact of amplicons embedded in one or more hydrogels occurs six or more times for thick tissue samples. In some embodiments, one or more amplicons can be contacted by 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 hours or less, 45 hours or less, 30 hours or less, 25 hours or less, 20 hours or less, 15 hours or less, 10 hours or less, 5 hours or less, or 2 hours or less. 【0058】 Samples prepared using this method can be analyzed by any of several different types of microscopy, such as optical microscopy (e.g., bright-field, tilt-illuminated, dark-field, phase-contrast, differential interference contrast, interference reflection, epifluorescence, confocal, etc.), laser microscopy, electron microscopy, and scanning probe microscopy. In some embodiments, non-transient computer-readable media are used to first convert raw images obtained through multiple rounds of microscopy of in-situ sequencing into decoded gene identity and spatial location, and then to analyze the per-cell composition of gene expression. 【0059】 SEDAL oligonucleotide primer In some embodiments, the disclosed method includes a third oligonucleotide and a fourth oligonucleotide. In certain embodiments, the third oligonucleotide is configured to decode a base, and the fourth oligonucleotide is configured to convert the decoded base into a signal. In some embodiments, the signal is a fluorescent signal. In an exemplary embodiment, contacting an amplicon embedded in one or more hydrogels having a barcode sequence with a pair of primers under conditions that allow ligation involves each of the third and fourth oligonucleotides ligating to form a stable product for imaging only if a perfect match occurs. In certain embodiments, the mismatch sensitivity of a ligase enzyme is used to determine the baseline sequence of a target nucleic acid molecule. 【0060】 The term "perfectly matched," when used in reference to double-stranded structures, means that the polynucleotide and / or oligonucleotide strands constituting the double-stranded structure form a double-stranded structure with each other such that all nucleotides in each strand undergo Watson-Crick base pairing with nucleotides in the other strand. The term "double-stranded" can be used to include, but is not limited to, pairings of deoxyinosine, nucleosides with 2-aminopurine bases, and nucleoside analogs such as peptide nucleic acids (PNAs). "Mismatch" in a double-stranded structure between two oligonucleotides means that a pair of nucleotides in the double-stranded structure cannot undergo Watson-Crick bonding. 【0061】 In some embodiments, the method includes multiple third oligonucleotides, including, but not limited to, five or more third oligonucleotides that hybridize to a target nucleotide sequence, e.g., eight or more, ten or more, twelve or more, fifteen or more, eighteen or more, twenty or more, twenty-five or more, thirty or more, thirty or more, or thirty or more. In some embodiments, the method of the present disclosure includes multiple third oligonucleotides, including, but not limited to, fifteen or more third oligonucleotides that hybridize to fifteen or more, e.g., twenty or more, thirty or more, forty or more, fifty or more, sixty or more, seventy or more, and up to eighty different first oligonucleotides, e.g., 20 or more, thirty or more, forty or more, fifty or more, sixty or more, seventy or more, and up to eighty different target nucleotide sequences. In some embodiments, the method includes, but is not limited to, a plurality of fourth oligonucleotides, comprising five or more fourth oligonucleotides, e.g., eight or more, ten or more, twelve or more, fifteen or more, eighteen or more, twenty or more, twenty-five or more, thirty or more, thirty or more, thirty or more. In some embodiments, the method of the present disclosure includes, but is not limited to, a plurality of fourth oligonucleotides, comprising fifteen or more fourth oligonucleotides, e.g., twenty or more, thirty or more, forty or more, fifty or more, sixty or more, seventy or more, and up to eighty different first oligonucleotides, hybridizing to fifteen or more, e.g., twenty or more, thirty or more, forty or more, fifty or more, sixty or more, seventy or more, and up to eighty different target nucleotide sequences. If one or more pairs specifically bind to each target nucleic acid, a plurality of oligonucleotide pairs can be used in the reaction. For example, two primer pairs can be used for one target nucleic acid to improve sensitivity and reduce variability. The objective is to detect multiple different target nucleic acids within a cell, for example, up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 25, 30, or even 40 or more different target nucleic acids. 【0062】 In certain embodiments, SEDAL comprises a ligase having activity inhibited by base mismatch, a third oligonucleotide, and a fourth oligonucleotide. In this context, the term “inhibited” refers to ligase activity reduced by approximately 20% or more, e.g., 25% or more, e.g., 50% or more, e.g., 75% or more, e.g., 90% or more, e.g., 95% or more, e.g., 99% or more, e.g., 100%. In some embodiments, the third oligonucleotide has a length of 5 to 15 nucleotides, but is not limited to these, and contains 5 to 13 nucleotides, 5 to 10 nucleotides, or 5 to 8 nucleotides. In some embodiments, the T of the third oligonucleotide m The temperature is room temperature (22-25°C). In some embodiments, the third oligonucleotide is degenerate or partially degenerate. In some embodiments, the fourth oligonucleotide has a length of 5-15 nucleotides, but is not limited to these, and contains 5-13 nucleotides, 5-10 nucleotides, or 5-8 nucleotides. In some embodiments, the T of the fourth oligonucleotide m The temperature is room temperature (22-25°C). After each cycle of the SEDAL corresponding to the base readout, the fourth oligonucleotide may be detached, which eliminates the accumulation of errors as sequencing progresses. In such embodiments, the fourth oligonucleotide is detached by formamide. 【0063】 In some embodiments, SEDAL involves washing the third and fourth oligonucleotides to remove unbound oligonucleotides, and then revealing the fluorescent product for imaging. In certain exemplary embodiments, one or more nucleotides and / or oligonucleotides described herein can be detected using detectable labels. In certain embodiments, one or more amplicons can be detected using detectable labels. Examples of detectable markers include various radioactive moieties, enzymes, prosthesis groups, fluorescent markers, luminescent markers, bioluminescent markers, metal particles, protein-protein binding pairs, protein-antibody binding pairs, etc. Examples of fluorescent proteins include, but are not limited to, yellow fluorescent protein (YFP), green fluorescent protein (GFP), cyanide fluorescent protein (CFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin, etc. Examples of bioluminescent markers include, but are not limited to, luciferases (e.g., bacteria, fireflies, click beetles, etc.), luciferin, aequorin, etc. Examples of enzyme systems with visually detectable signals include, but are not limited to, galactosidases, glucolimidases, phosphatases, peroxidases, and cholinesterases. Identifiable markers include: 125 I, 35 S, 14 C, or 3 It also contains radioactive compounds such as H. Identifiable markers are commercially available from various sources. 【0064】 Fluorescent labeling, and their attachment to nucleotides and / or oligonucleotides, is described in Haugland's 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, editor, Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991), and Wetmur, Critical Reviews in Numerous reports describe this invention, including Biochemistry and Molecular Biology, 26:227-259 (1991). Specific methodologies applicable to the present invention are disclosed in the references of U.S. Patents No. 4,757,141, No. 5,151,507, and No. 5,091,519. In one embodiment, one or more fluorescent dyes are used as labels for a labeled target sequence, as disclosed, for example, by U.S. Patent No. 5,188,934 (4,7-dichlorofluorescein dye), U.S. Patent No. 5,366,860 (spectrally degradable rhodamine dye), U.S. Patent No. 5,847,162 (4,7-dichlororhodamine dye), U.S. Patent No. 4,318,846 (ether-substituted fluorescein dye), U.S. Patent No. 5,800,996 (energy transfer dye), Lee et al., U.S. Patent No. 5,066,580 (xanthine dye), U.S. Patent No. 5,688,648 (energy transfer dye), etc. Labeling can be performed using quantum dots as disclosed in U.S. Patents and Publications No. 6,322,901, 6,576,291, 6,423,551, 6,251,303, 6,319,426, 6,426,513, 6,444,143, 5,990,479, 6,207,392, 2002 / 0045045, and 2003 / 0017264. As used herein, the term “fluorescent labeling” includes a signaling moiety that transmits information through the fluorescent absorption and / or emission properties of one or more molecules. Such fluorescent properties include fluorescence intensity, fluorescence lifetime, emission spectral properties, energy transfer, and the like. 【0065】 Commercially available fluorescent nucleotide analogs that readily integrate 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, TEXAS RED®-5-dUTP, CASCADE BLUE®-7-dUTP, BODIPY TMFL-14-dUTP, BODIPY TMR-14-dUTP, BODIPY TMTR-14-dUTP, RHODAMINE GREEN®-5-dUTP, OREGON GREEN®-488-5-dUTP, and 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, ALEXA FLUOR(TM) 568-5-dUTP, ALEXA FLUOR(TM) 594-5-dUTP, ALEXA-FLUOR(TM) 546-14-dUTP, Fluorescein-12-UTP, Tetramethylrhodermine-6-UTP, TEXAS RED(TM)-5-UTP, mCHERRY, CASCADE BLUE(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 Examples include Probes, Inc. (Eugene, Oregon). Protocols for the custom synthesis of nucleotides with other fluorophores (see Henegariu et al. (2000) Nature Biotechnol, 18:345) are known in the art. 【0066】 Other fluorophores available for post-synthesis attachment include, but are not limited to, ALEXA FLUOR® 350, ALEXA FLUOR® 532, and ALEXA FLUOR(trademark) 546, ALEXA FLUOR(trademark) 568, ALEXA FLUOR(trademark) 594, ALEXA FLUOR(trademark) 647, BODIPY 493 / 503, BODIPY FL, BODIPY R6G, BODIPY 530 / 550, BODIPY TMR, BODIPY 558 / 568, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, Cascade Blue, Cascade Yellow, Dansil, Lissamine Rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Examples include Blue, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Tetramethylrhodamine, Texas Red (available from Molecular Probes, Inc., Eugene, Oregon), Cy2, Cy3.5, Cy5.5, and Cy7 (Amersham Biosciences, Piscataway, New Jersey). 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), and APC-Alexa dyes. 【0067】 Metallic silver or gold particles can be used to enhance the signal from fluorescently labeled nucleotide and / or oligonucleotide sequences (Lakowicz et al. (2003) Bio Techniques 34:62). 【0068】 Biotin, or its derivatives, may also be used as labels on nucleotide and / or oligonucleotide sequences and subsequently conjugated by a detectably labeled avidin / streptavidin derivative (e.g., hycoerythrin-conjugated streptavidin) or a detectably labeled anti-biotin antibody. Digoxigenin may be incorporated as a label and subsequently conjugated by a detectably labeled anti-digoxigenin antibody (e.g., fluorescein-conjugated anti-digoxigenin). Aminoaryl-dUTP residues may be incorporated into oligonucleotide sequences and subsequently conjugated to N-hydroxysuccinimide (NHS)-derivativeized fluorescent dyes. In general, any member of a conjugation pair may be incorporated into a detection oligonucleotide, provided that a detectably labeled conjugation partner can conjugate to it to enable detection. As used herein, the term antibody refers to any class of antibody molecules, such as Fab, or any sub-fragment thereof. 【0069】 Other suitable labels for oligonucleotide sequences include fluorescein (FAM), digoxigenin, dinitrophenol (DNP), dansyl, biotin, bromodeoxyuridine (BrdU), hexahistidine (6×His), and phospho-amino acids (e.g., P-tyr, P-ser, P-thr). In one embodiment, hapten / antibody pairs under biotin / α-biotin, digoxigenin / α-digoxigenin, dinitrophenol (DNP) / α-DNP, and 5-carboxyfluorescein (FAM) / α-FAM are used for detection, and each antibody is derivatized with a detectable label. 【0070】 In certain exemplary embodiments, nucleotide and / or oligonucleotide sequences may be indirectly labeled with haptens subsequently bound by a scavenger, as disclosed, for example, in U.S. Patents 5,344,757, 5,702,888, 5,354,657, 5,198,537, and 4,849,336, PCT Publication WO91 / 17160, etc. Many different hapten scavenger pairs are available. Exemplary haptens include, but are not limited to, biotin, desbiotin, and other derivatives, dinitrophenol, dansyl, fluorescein, CY5, digoxigenin, and others. For biotin, the scavenger may be avidin, streptavidin, or an antibody. Antibodies can be used as scavengers for other haptens (many dye-antibody pairs are commercially available, e.g., Molecular Probes, Eugene, Oregon). 【0071】 cell The methods disclosed herein include methods for in situ gene sequencing of target nucleic acids in cells within intact tissue. In certain embodiments, the cells are present in a population of cells. In certain other embodiments, the population of cells includes, but is not limited to, several cell types, including excitatory neurons, inhibitory neurons, and non-neuronal cells. Cells for use in the assays of the present invention may be organisms, single cell types derived from organisms, or mixtures of cell types. These include naturally occurring cells and cell populations, genetically engineered cell lines, cells derived from transgenic animals, and so on. Virtually any cell type and size can be accommodated. 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 from naturally occurring tissues, such as blood, liver, pancreas, nerve tissue, bone marrow, and skin. Some tissues may be divided into monodisperse suspensions. Alternatively, the cells may be cultured populations, for example, cultures derived from complex populations, cultures derived from single cell types in which cells have differentiated into numerous lineages, or cells that respond differentially to stimuli. 【0072】 Cell types that can be used in the present invention include stem cells and progenitor cells, such as embryonic stem cells, hematopoietic stem cells, mesenchymal stem cells, and apical cells; endothelial cells, muscle cells, cardiomyocytes, smooth muscle and skeletal muscle cells, mesenchymal cells, epithelial cells; hematopoietic cells such as lymphocytes including T cells such as Th1 T cells, Th2 T cells, ThO T cells, and cytotoxic T cells; B cells, pre-B cells, etc.; monocytes; dendritic cells; neutrophils; and macrophages; natural killer cells, mast cells, etc.; adipocytes, cells involved in specific organs such as the thyroid gland, endocrine glands, pancreas, and brain, such as neurons, glial cells, astrocytes, and dendritic cells, as well as genetically modified cells thereof. Hematopoietic cells may be associated with inflammatory processes and autoimmune diseases, endothelial cells, smooth muscle cells, cardiomyocytes, etc. may be associated with cardiovascular diseases, and almost any type of cell may be associated with tumors such as sarcomas, cancers, and lymphomas, liver diseases with hepatocytes, and kidney diseases with renal cells. 【0073】 Cells can also be different types of transformed or neoplastic cells, such as sarcomas of different cellular origins or lymphomas of different cell types. The American Type Culture Collection (Manassas, Virginia) has collected and made available over 4,000 cell lines from more than 150 different species, and over 950 cancer cell lines, including 700 human cancer cell lines. The National Cancer Institute has accumulated clinical, biochemical, and molecular data from a large panel of human tumor cell lines, which are available from ATCC or NCI (Phelps et al.). al. (1996) Journal of Cellular Biochemistry Supplement 24:32-91). This may include a number of cell lines that are spontaneously derived or selected from individual cell lines for desired growth or response characteristics, and that originate from similar tumor types but from different patients or sites. 【0074】 Cells can be non-adherent cells, such as blood cells including monocytes, T cells, and B cells, or tumor cells, or adherent cells, such as epithelial cells, endothelial cells, and nerve cells. To profile adherent cells, they can be detached from the substrate to which they are attached and from other cells in a manner that maintains their ability to identify and bind probe molecules. 【0075】 Such cells can be obtained from individuals using various techniques known in the art, such as extraction, washing, flushing, surgical dissection, etc., from various tissues, e.g., blood, bone marrow, solid tissues (e.g., solid tumors), and ascites fluid. Cells can be obtained from fixed or unfixed, fresh or frozen, whole or dispersed samples. Dispersion of tissues can be achieved mechanically or enzymatically using known techniques. 【0076】 imaging The disclosed method includes imaging amplicons embedded in one or more hydrogels using any of several different types of microscopy, such as confocal microscopy, two-photon microscopy, bright-field microscopy, intact tissue expansion microscopy, and / or CLARITY® optimized light sheet microscopy (COLM). 【0077】 Bright-field microscopy is the simplest of all optical microscopy techniques. Sample illumination is via transmitted white light, i.e., irradiated from below and observed from above. Limitations include low contrast and low apparent resolution for most biological samples due to blurring of out-of-focus material. The simplicity of the technique and the minimal sample preparation required are major advantages. 【0078】 In tilt illumination microscopy, the specimen is illuminated from the side. This gives the image a three-dimensional appearance and allows for the highlighting of features that would otherwise be invisible. A more recent technique based on this method is Hoffmann modulation contrast, a system found on inverted microscopes used for cell culture. While tilt illumination suffers from the same limitations as bright-field microscopy (low contrast and low apparent resolution for many biological specimens due to blurring of out-of-focus material), it can highlight structures that would otherwise be invisible. 【0079】 Dark-field microscopy is a technique for improving the contrast of unstained, transparent specimens. Dark-field illumination uses carefully aligned light sources to minimize the amount of directly transmitted (unscattered) light entering the image plane, collecting only the light scattered by the specimen. Dark-field dramatically improves image contrast (especially for transparent objects) while requiring little instrument setup or specimen preparation. However, this technique continues to suffer from low light levels in the final images of many biological specimens and is affected by low apparent resolution. 【0080】 Phase contrast is an optical microscope illumination technique that converts the phase shift of light passing through a transparent sample into a change in brightness in the image. In other words, phase contrast shows the difference in refractive index as a difference in contrast. The phase shift itself is invisible to the human eye, but it becomes visible when shown as a change in brightness. 【0081】 In differential interference contrast (DIC) microscopy, differences in optical density will be displayed as differences in relief. This system consists of special prisms (normal-ski prism, Wollaston prism) in a condenser that split the light into ordinary and extraordinary rays. The spatial difference between the two rays is minimal (less than the maximum resolution of the objective lens). After passing through the specimen, the rays are recombined by similar prisms in the objective lens. In a homogeneous specimen, there is no difference between the two rays, and no contrast is generated. However, near the refractive boundary (e.g., the nucleus in the cytoplasm), the difference between the ordinary and extraordinary rays will generate relief in the image. Differential interference contrast requires a polarized light source, and two polarizing filters must be fitted into the optical path, one below the condenser (polarizer) and the other above the objective lens (analyzer). 【0082】 Another microscopy technique that uses interference is interference reflection microscopy (also known as reflection interference contrast, or RIC). This is used to examine the adhesion of cells to a glass surface, using a narrow range of reflected wavelength polarization whenever there is an interface between two materials with different refractive indices. Whenever cells are adhered to the glass surface, the reflected light from the glass and the reflected light from the adherent cells will interfere. If cells are not adhered to the glass, there will be no interference. 【0083】 A fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence instead of, or in addition to, reflection and absorption, to study the properties of organic or inorganic materials. In fluorescence microscopy, the sample is illuminated with light of a wavelength that excites fluorescence in the sample. The fluorescence light, which is usually a longer wavelength than the illumination, is then imaged through the microscope objective lens. This technique can use two filters: an illumination (or excitation) filter that ensures the illumination is nearly monochromatic and of the correct wavelength, and a second emission (or barrier) filter that ensures that neither of the excitation light sources reaches the detector. Alternatively, both of these functions can be achieved by a single two-color 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 uses optical sections to obtain better resolution of the fluorescence image. 【0084】 Confocal microscopy uses point illumination and a pinhole in an optically joined plane in front of the detector to eliminate out-of-focus signals. Because only light produced by fluorescence very close to the focal plane is detectable, the optical resolution of the image is far superior to that of wide-field microscopy, particularly in the depth direction of the sample. However, this increase in resolution comes at the cost of reduced signal intensity, as much of the light from the sample's fluorescence is blocked by the pinhole, often requiring prolonged exposure. Since only one point in the sample is illuminated at a time, 2D or 3D imaging requires scanning across a regular raster (i.e., a rectangular pattern of parallel scanning lines) within the specimen. The achievable thickness of the focal plane is, in most cases, defined by the wavelength of the light used divided by the numerical aperture of the objective lens, but is also defined by the optical properties of the specimen. Possible thin optical sections make these types of microscopes particularly excellent for 3D imaging and surface profiling of specimens. COLM offers an alternative microscopy technique for fast 3D imaging of large, clarified specimens. COLM allows for the investigation of large immunostained tissues, improving acquisition speed and resulting in higher quality data. 【0085】 In single-plane illumination microscopy (SPIM), also known as light-sheet microscopy, only fluorophores within the focal plane of the detection objective lens are illuminated. A light-sheet is a beam of light sighted in one direction and focused in the other. Because fluorophores are not excited outside the detector's focal plane, this method also provides unique optical sections. Furthermore, compared to conventional microscopy, the light-sheet method exhibits reduced photobleaching and lower phototoxicity, and often allows for significantly more scans per specimen. By rotating the specimen, this technique can image virtually any plane with numerous views acquired from different angles. However, at any angle, only relatively shallow sections of the specimen are imaged with high resolution, while deeper areas appear increasingly blurred. 【0086】 Super-resolution microscopy is a form of optical microscopy. Due to the diffraction of light, the resolution of conventional optical microscopy is limited, as described by Ernst Abbe in 1873. A good approximation of the achievable resolution is the FWHM (full width at maximum radius) of the point diffusion function, and accurate wide-field microscopes with high aperture values ​​and visible light typically achieve a resolution of about 250 nm. Super-resolution techniques enable the acquisition of images with a resolution higher than the diffraction limit. They are classified into two broad categories: “true” super-resolution techniques, which capture information contained in evanescent waves, and “functional” super-resolution techniques, which use experimental techniques and known limits on the material being imaged to reconstruct a super-resolution image. 【0087】 Laser microscopy utilizes laser illumination sources in various forms of microscopy. For example, laser microscopy focused on biological applications uses ultrashort wave pulsed lasers or femtosecond lasers in several techniques, including nonlinear microscopy, saturation microscopy, and multiphoton fluorescence microscopy such as two-photon excitation microscopy (a fluorescence imaging technique that enables imaging of biological tissue to very high depths of up to 1 millimeter). 【0088】 Electron microscopy (EM) uses electron beams to illuminate a specimen and generate a magnified image. Electron microscopes have greater resolution than optical power microscopes because electrons have wavelengths approximately 100,000 times shorter than visible light (photons). They can achieve resolutions exceeding 50 pm and magnifications up to approximately 10,000,000x, whereas typical non-focused optical microscopes are limited by diffraction to a resolution of approximately 200 nm and a useful magnification below 2000x. Electron microscopes use electrostatic and electromagnetic "lenses" to control and focus the electron beam to form an image. These lenses are similar to, but different from, the glass lenses in optical microscopes that focus light on or through the specimen to form a magnified image. Electron microscopes are used to observe a wide range of biological and inorganic specimens, including microorganisms, cells, macromolecules, biopsy samples, metals, and crystals. Industrially, electron microscopes are often used in quality control and failure analysis. Examples of electron microscopy include transmission electron microscopy (TEM), scanning electron microscopy (SEM), backscattered electron microscopy (REM), scanning transmission electron microscopy (STEM), and low-voltage electron microscopy (LVEM). 【0089】 Scanning probe microscopy (SPM) is a branch of microscopy that uses a physical probe to scan a specimen and form an image of its surface. The surface image is acquired by mechanically moving the probe through a raster scan of the specimen and, line by line, recording the probe-surface interaction as a function of position. Examples of SPM include atomic energy microscopy (ATM), ballistic electron emission microscopy (BEEM), chemical force microscopy (CFM), conductive atomic energy microscopy (C-AFM), electrochemical scanning tunneling microscopy (ECSTM), electrostatic force microscopy (EFM), fluid force microscopy (FluidFM), force modulation microscopy (FMM), function-oriented scanning probe microscopy (FOSPM), Kelvin probe force microscopy (KPFM), magnetic force microscopy (MFM), magnetic resonance force microscopy (MRFM), near-field scanning optical microscopy (NSOM) (or SNOM), scanning near-field optical microscopy, SNOM, piezoelectric response force microscopy (PFM), PSTM, photon scanning tunneling microscopy (PSTM), PTMS. Examples include photothermal microscopy / microscopy (PTMS), scanning capacitance microscopy (SCM), scanning electrochemical microscopy (SECM), scanning gate microscopy (SGM), scanning Hall probe microscopy (SHPM), scanning ion conduction microscopy (SICM), spin polarization scanning tunneling microscopy (SPSM), scanning diffusion resistance microscopy (SSRM), scanning thermal microscopy (SThM), scanning tunneling microscopy (STM), scanning tunneling potentiometer (STP), scanning voltage microscopy (SVM), and synchrotron X-ray scanning tunneling microscopy (SXSTM). 【0090】 Expansion microscopy (exM) enables imaging of thick, preserved specimens with an azimuthal resolution of approximately 70 nm. ExM avoids the optical diffraction limit by physically expanding the biological specimen before imaging, thus bringing sub-diffraction-limited structures into a size range visible by conventional diffraction-limited microscopy. ExM allows imaging of biological specimens at the voxel rate of diffraction-limited microscopy, but with the voxel size of super-resolution microscopy. The expanded specimen is transparent, and since the expanded material is >99% water, its refractive index matches that of water. The technique of expansion microscopy is known in the art, for example, as disclosed in Gao et al., Q&A: Expansion Microscopy, BMC Biol. 2017;15:50. 【0091】 Screening method The method disclosed herein also provides a method for screening candidate drugs to determine whether a candidate drug modulates gene expression of nucleic acids in cells within intact tissue, the method comprising (a) contacting immobilized and permeabilized intact tissue with at least a pair of oligonucleotide primers under conditions that enable specific hybridization, wherein the primer pair comprises a first oligonucleotide and a second oligonucleotide, each of the first and second oligonucleotides comprising a first complementary region and a second complementary region The second oligonucleotide further comprises a barcode sequence, the first complementary region of the first oligonucleotide being complementary to the first portion of the target nucleic acid, the second complementary region of the first oligonucleotide being complementary to the first complementary region of the second oligonucleotide, the third complementary region of the first oligonucleotide being complementary to the third complementary region of the second oligonucleotide, the second complementary region of the second oligonucleotide being complementary to the second portion of the target nucleic acid, and the first complementary region of the first oligonucleotide (b) Contacting the second oligonucleotide having a sexual region adjacent to the second complementary region of the second oligonucleotide; (c) Adding a ligase to ligate the second oligonucleotide and generate a closed nucleic acid ring; (d) Performing rolling circle amplification in the presence of nucleic acid molecules, using the second oligonucleotide as a template and the first oligonucleotide as a primer for polymerase to form one or more amplicons; (e) Embedding one or more amplicons in the presence of a hydrogel subunit to form amplicons embedded in one or more hydrogels; (e) Contacting the amplicons embedded in one or more hydrogels having a barcode sequence with a pair of primers under conditions that allow ligation, the pair of primers comprising a third oligonucleotide and a fourth oligonucleotide, and ligation occurs only when both the third oligonucleotide and the fourth oligonucleotide ligate to the same amplicon; and (f) Repeating step (e) many times.(g) imaging amplicons embedded in one or more hydrogels to determine the in-situ gene sequencing of a target nucleic acid in cells within intact tissue; and (h) detecting the level of gene expression of the target nucleic acid, wherein the change in the level of expression of the target nucleic acid in the presence of at least one candidate drug compared to the level of expression of the target nucleic acid in the absence of at least one candidate drug indicates that at least one candidate drug modulates gene expression of the nucleic acid in cells within intact tissue. Such a screening method includes the steps of STARmap provided herein. 【0092】 In some embodiments, detection includes performing flow cytometry, sequencing, probe binding and electrochemical detection, pH changes, catalysis induced by enzymes bound to DNA tags, quantum entanglement, Raman spectroscopy, terahertz wave techniques, and / or scanning electron microscopy. In certain embodiments, flow cytometry is mass cytometry or fluorescence-activated flow cytometry. In some other embodiments, detection includes performing microscopy, scanning mass spectrometry, or other imaging techniques described herein. In such embodiments, detection includes determining a signal, for example, a fluorescence signal. 【0093】 As used interchangeably herein, “test agent,” “candidate agent,” and grammatical equivalents mean any molecule (e.g., protein (including proteins, polypeptides, and peptides herein), small molecule (i.e., 5–1000 Da, 100–750 Da, 200–500 Da, or less than 500 Da in size), or organic or inorganic molecule, polysaccharide, polynucleotide, etc.) that is tested for activity in the assay of interest. 【0094】 Various different candidate drugs can be screened using the method described above. Candidate drugs include a number of chemical classes, for example, small organic compounds having molecular weights greater than 50 daltons (e.g., at least about 50 Da, at least about 100 Da, at least about 150 Da, at least about 200 Da, at least about 250 Da, or at least about 500 Da) and less than about 20,000 daltons, less than about 10,000 daltons, less than about 5,000 daltons, or less than about 2,500 daltons. For example, in some embodiments, preferred candidate agents are organic compounds having molecular weights in the range of about 500 Da to about 20,000 Da, for example, about 500 Da to about 1000 Da, about 1000 Da to about 2000 Da, about 2000 Da to about 2500 Da, about 2500 Da to about 5000 Da, about 5000 Da to about 10,000 Da, or about 10,000 Da to about 20,000 Da. 【0095】 Candidate drugs may contain functional groups necessary for structural interactions with proteins, such as hydrogen bonding, and may contain at least two of the following: amine, carbonyl, hydroxyl, or carboxyl groups, or functional chemical groups. Candidate drugs may contain cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate drugs are also found in biomolecules including peptides, sugars, fatty acids, steroids, purines, pyrimidines, their derivatives, structural analogs, or combinations thereof. 【0096】 Candidate drugs can be obtained from a wide variety of sources, including libraries of synthetic or natural compounds. For example, numerous means are available for the random and directed synthesis of a wide variety of organic compounds and biomolecules, including the expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or readily produced. In addition, libraries and compounds produced naturally or synthetically can be readily modified through conventional chemical, physical, and biochemical means and used to produce combination libraries. Known pharmacological agents may be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, and amidation to produce structural analogs. Furthermore, screening can be directed to known pharmacologically active compounds and their chemical analogs, or to novel drugs with unknown properties, such as those created by rational drug design. 【0097】 In one embodiment, the candidate modulator is a synthetic compound. Any number of techniques are available for the random and directed synthesis of a wide variety of organic compounds and biomolecules, including the expression of randomized oligonucleotides. For example, see WO94 / 24314, expressly incorporated herein by reference, for methods for generating novel compounds, including random chemical and enzymatic methods. 【0098】 In another embodiment, candidate agents are provided as a library of natural compounds in the form of bacterial, fungal, plant, and animal extracts that are available or readily produced. In addition, libraries and compounds produced naturally or synthetically are readily modified through conventional chemical, physical, and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, including enzymatic modification, to produce structural analogues. 【0099】 In one embodiment, the candidate drug comprises a protein (antibody, antibody fragment (i.e., a fragment containing an antigen-binding region, a single-chain antibody, etc.)), nucleic acid, and chemical moiety. In one embodiment, the candidate drug is a naturally occurring protein or a fragment of a naturally occurring protein. Therefore, for example, a cell extract containing a protein, or a random or targeted digest of a proteinaceous cell extract, may be tested. In this way, libraries of prokaryotic and eukaryotic proteins can be prepared for screening. Other embodiments include libraries of bacterial, fungal, viral, and mammalian proteins (e.g., human proteins). 【0100】 In one embodiment, the candidate drug is an organic moiety. In this embodiment, the candidate drug is synthesized from a set of chemically modifiable substrates, as generally described in WO94 / 24314. "Chemically modified" as used herein includes conventional chemical reactions as well as enzymatic reactions. These substrates generally include, but are not limited to, alkyl groups (including alkanes, alkenes, alkynes, and heteroalkyls), aryl groups (including allenes and heteroaryls), alcohols, ethers, amines, aldehydes, ketones, acids, esters, amides, cyclic compounds, heterocyclic compounds (including purines, pyrimidines, benzodiazepines, beta-lactams, tetracillins, cephalosporins, and carbohydrates), steroids (including estrogens, androgens, cortisones, ecodysones, etc.), alkaloids (including ergot, vinca, curelle, pyrrolidine, and mitomycin), organometallic compounds, heteroatom-supported compounds, amino acids, and nucleosides. A chemical reaction (including an enzymatic reaction) may be carried out on a portion of the surface to form a new substrate or candidate drug that can then be tested using the present invention. 【0101】 Devices and Systems The method also includes devices for carrying out embodiments of this method. These devices may include, for example, an imaging chamber, an electrophoresis apparatus, a flow chamber, a microscope, a needle, tubing, and a pump. 【0102】 This disclosure also provides a system for carrying out the method. The system may comprise one or more of the modules described herein, such as a power supply, a refrigeration unit, a waste disposal unit, a heating unit, a pump, etc. The system may also comprise any of the reagents described herein, such as imaging buffer, washing buffer, strip buffer, Nissl and DAPI solutions. The system according to a particular embodiment may also comprise a microscope and / or associated imaging equipment, such as camera components, digital imaging components, and / or image acquisition equipment, a computer processor configured to collect images according to one or more user inputs, etc. 【0103】 As discussed above, the system described herein comprises a fluid device having an imaging chamber and a pump, and a processor unit configured to perform a method for in situ gene sequencing of target nucleic acids in cells within intact tissue as described herein. In some embodiments, the system enables the automation of STARmap, which is a process described herein, including, but not limited to, repeated rounds of probe hybridization with DNA embedded in a gel, ligation of fluorescently labeled oligonucleotides on these probes, washing away excess probe, imaging, and detaching the probes for sequencing in the next round. In some embodiments, the system may enable continuous operation. In some embodiments, the system comprises an imaging chamber for flowing sequencing chemicals involved in in situ DNA sequencing on a sample. In some embodiments, the fluid and pump system controls the delivery of the sequencing chemicals to the sample. 【0104】 The buffer solution can be added / removed / recirculated / replaced by one or more ports and, optionally, by the use of tubing, pumps, valves, or any other suitable fluid handling and / or fluid operating equipment, e.g., tubing that is detachably or permanently attached to one or more components of the device. For example, a first tubing having first and second ends can be attached to a first port, a second tubing having first and second ends can be attached to a second port, the first end of the first tubing can be attached to a first port and the second end of the first tubing can be operably connected to a receptacle, e.g., a cooling unit, heating unit, filtration unit, waste receptacle, etc., the first end of the second tubing can be attached to a second port and the second end of the second tubing can be operably connected to a receptacle, e.g., a cooling unit, ice beaker, filtration unit, waste receptacle, etc. 【0105】 In some embodiments, the system includes a non-transient computer-readable storage medium containing instructions, which, when executed by a processor unit, cause the processor unit to control the delivery of a chemical substance and synchronize this process with a microscope. In some embodiments, the non-transient computer-readable storage medium includes instructions, when executed by a processor unit, causing the processor unit to measure an optical signal. 【0106】 Utility The devices, methods, and systems described herein find several uses in the art, for example, in biomedical research and / or clinical diagnostics. 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 diagnostics, applications include, but are not limited to, detecting genetic markers such as disease, immune response, bacterial or viral DNA / RNA in patient samples. Examples of the advantages of the methods described herein include efficiency, which provides a much faster rate than existing microarray or sequencing techniques, with final data obtained from raw samples in just 3 or 4 days, high multiplexing (up to 1000 genes), single-cell and single-molecule sensitivity, conserved tissue morphology, and / or a high signal-to-noise ratio with a low error rate. 【0107】 In certain embodiments, STARmap can be applied to studies of molecularly defined cell types and regulated gene expression in the mouse visual cortex and may be extendable to larger 3D tissue blocks to visualize the short-range and long-range spatial configuration of cortical neurons at a volume scale that was previously inaccessible. In some embodiments, the methods disclosed herein may be adapted to DNA-conjugated antibodies for highly multiplexed protein detection. 【0108】 The devices, methods, and systems of the present invention can also be generalized to study several heterogeneous cell populations in diverse tissues. Without being constrained by any scientific theory, the brain presents a particular challenge well suited to STARmap analysis. For example, polymorphic regulated gene (ARG) expression observed across different cell types is likely to depend on both endogenous cell biological properties (such as the expression of signaling pathway components) and extrinsic properties such as the neural circuit biostructure that transmits external sensory information to different cells (in this case, the visual cortex). In such cases, in situ transcriptotomics, exemplified by STARmap, can effectively link imaging-based molecular information with anatomical and activity information, and thus elucidate brain function and dysfunction. 【0109】 The devices, methods, and systems disclosed herein allow for the removal of cellular components, such as lipids that typically provide structural support but hinder the visualization of subcellular proteins and molecules, while simultaneously preserving the three-dimensional constructs of cells and tissues, as the sample is crosslinked to a hydrogel that physically supports the tissue's superstructure. This removal substantially makes the interior of the biological sample permeable to light and / or macromolecules, enabling microscopic visualization of the interior of the sample, such as cells and subcellular structures, without the time-consuming destructive cutting of the tissue. This procedure is faster than procedures commonly used in the art, as sweeping and permeabilization, typically performed in separate steps, can be combined in a single step of removing cellular components. In addition, for comprehensive analysis, the sample can be repeatedly stained, left unstained, and restained with other reagents. Further functionalization with polymerizable acrylamide moieties allows for the covalent immobilization of amplicons within a polyacrylamide network at numerous sites. 【0110】 For example, the devices, methods, and systems may be used to assess, diagnose, or monitor a disease. As used herein, “diagnosis” generally includes predicting susceptibility to a disease or disorder in a subject, determining whether a subject is currently suffering from a disease or disorder, predicting the prognosis of a subject suffering from a disease or disorder (e.g., identifying a cancerous state, the stage of cancer, the likelihood of the patient dying from cancer), predicting the subject’s responsiveness to treatment for the disease or disorder (e.g., positive response, negative response, or no response to allogeneic hematopoietic stem cell transplantation, chemotherapy, radiotherapy, antibody therapy, small molecule compound therapy, etc.), and using therapeutic measurements (e.g., monitoring the subject’s condition to provide information regarding the effectiveness or efficacy of treatment). For example, a biopsy may be prepared from cancerous tissue and microscopically analyzed to determine the type of cancer, the extent to which the cancer has developed, and whether the cancer will respond to therapeutic intervention. 【0111】 This device, method, and system also provide a useful technique for screening candidate therapeutic agents for their effects on tissues or diseases. For example, a subject, such as a mouse, rat, dog, primate, or human, may be brought into contact with a candidate drug, or its organs or biopsies may be prepared by this method, and prepared specimens microscopically analyzed for one or more cellular or tissue parameters. Parameters are quantifiable components of cells or tissues, in particular components that can be accurately measured by a high-throughput system, preferably. Parameters may be any cellular component or cell product, including cell surface determinants, receptors, proteins or their three-dimensional structure or post-translational modifications, lipids, carbohydrates, organic or inorganic molecules, nucleic acids, such as mRNA, DNA, etc., or portions derived from such cellular components, or combinations thereof. Most parameters will provide quantitative readouts, but in some cases semi-quantitative or qualitative results may be acceptable. Readouts may include a single determined value, or may include a mean, median, or variance, etc. Characteristically, the range of parameter readout values ​​will be obtained for each parameter from multiple identical assays. Variableity is expected, and the range of values ​​for each of the set of test parameters will be obtained using standard statistical methods, along with common statistical methods used to provide a single value. Thus, for example, one such method may include detecting cell viability, tissue angiogenesis, the presence of immune cell infiltration, or efficacy in altering disease progression. In some embodiments, the screening includes comparing the analyzed parameter(s) with those from a control or reference sample, e.g., a similarly prepared sample from an object that has not been in contact with the candidate drug. Target candidate drugs for screening include known and unknown compounds encompassing many chemical classes, primarily organic molecules, inorganic molecules, gene sequences, etc., which may include organometallic molecules. Target candidate drugs for screening also include nucleic acids, e.g., 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. Evaluation of tissue samples using this method may include, for example, gene analysis, transcriptional analysis, genomic analysis, proteomic analysis, and / or metabolic analysis. 【0112】 This device, method, and system also achieve subcellular resolution. For example, it can be used to visualize the distribution of genetically encoded markers throughout a tissue, such as chromosomal abnormalities (inversion, replication, translocation, etc.), loss of heterozygosity, the presence of alleles of genes indicating a predisposition to disease or good health, potential responsiveness to therapy, and ancestry. Such detection can be used, for example, in the diagnosis and monitoring of the aforementioned diseases, in personalized medicine, and in paternity studies. 【0113】 A database of analytical information can be accumulated. These databases may include results from known cell types, references from analyses of cells treated under specific conditions, etc. A data matrix can be generated, where each point in the data matrix corresponds to a readout from a cell, and the data for each cell may include readouts from numerous labels. The readouts may be the mean, median, or variance, or other statistically or mathematically derived values ​​related to the measurement. The output readout information can be further refined by directly comparing it with the corresponding reference readout. The absolute values ​​obtained for each output under identical conditions represent the variability inherent in the biological system and reflect both individual cell variability and inter-individual variability. 【0114】 Examples of non-limiting aspects of this disclosure The aspects of the subject matter described above (including embodiments) may be useful on their own or in combination with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the present disclosure numbered 1 to 89 are provided below. As will be apparent to those skilled in the art upon reading the present disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to support all such combinations of aspects, and is not limited to the combinations of aspects expressly provided below. 1. A method for determining the in-situ gene sequence of a target nucleic acid in cells within intact tissue, (a) Contacting immobilized and permeabilized intact tissue with at least one pair of oligonucleotide primers under conditions that enable specific hybridization, The primer pair includes a first oligonucleotide and a second oligonucleotide. Each of the first oligonucleotide and the second oligonucleotide comprises a first complementary region, a second complementary region, and a third complementary region, and the second oligonucleotide further comprises a barcode sequence. The first complementary region of the first oligonucleotide is complementary to the first portion of the target nucleic acid, the second complementary region of the first oligonucleotide is complementary to the first complementary region of the second oligonucleotide, the third complementary region of the first oligonucleotide is complementary to the third complementary region of the second oligonucleotide, the second complementary region of the second oligonucleotide is complementary to the second portion of the target nucleic acid, the first complementary region of the first oligonucleotide is adjacent to the second complementary region of the second oligonucleotide, (b) adding ligase to ligate the second oligonucleotide and generate a closed nucleic acid ring, (c) Performing rolling circle amplification in the presence of nucleic acid molecules, comprising forming one or more amplicons using a second oligonucleotide as a template and a first oligonucleotide as a primer for polymerase, (d) Embedding one or more amplicons in the presence of hydrogel subunits to form amplicons embedded in one or more hydrogels, (e) Contacting an amplicon embedded in one or more hydrogels having a barcode sequence with a pair of primers under conditions that enable ligation, wherein the pair of primers comprises a third oligonucleotide and a fourth oligonucleotide, and ligation occurs only when both the third and fourth oligonucleotides ligate to the same amplicon. (f) Repeat step (e) many times, (g) A method comprising imaging amplicons embedded in one or more hydrogels to determine the in-situ gene sequence of a target nucleic acid in cells within intact tissue. 2. The method according to embodiment 1, wherein a pair of primers are denatured by heating before contact with the sample. 3. The method according to embodiment 1 or 2, wherein the cells are present within a group of cells. 4. The method according to embodiment 3, wherein the cell population includes multiple cell types. 5. The method according to any one of embodiments 1 to 4, comprising contacting immobilized and permeabilized intact tissue to hybridize a pair of primers to the same target nucleic acid. 6. The method according to any one of embodiments 1 to 5, wherein the target nucleic acid is RNA. 7. The method according to embodiment 6, wherein RNA is mRNA. 8. The method according to any one of embodiments 1 to 5, wherein the target nucleic acid is DNA. 9. The method according to any one of embodiments 1 to 8, wherein the second oligonucleotide comprises a padlock probe. 10. The method according to any one of embodiments 1 to 9, wherein the first complementary region of the first oligonucleotide has a length of 19 to 25 nucleotides. 11. The method according to any one of embodiments 1 to 10, wherein the second complementary region of the first oligonucleotide has a length of 6 nucleotides. 12. The method according to any one of embodiments 1 to 11, wherein the third complementary region of the first oligonucleotide has a length of 6 nucleotides. 13. The method according to any one of embodiments 1 to 12, wherein the first complementary region of the second oligonucleotide has a length of 6 nucleotides. 14. The method according to any one of embodiments 1 to 13, wherein the second complementary region of the second oligonucleotide has a length of 19 to 25 nucleotides. 15. The method according to any one of embodiments 1 to 14, wherein the third complementary region of the second oligonucleotide has a length of 6 nucleotides. 16. The method according to any one of embodiments 1 to 15, wherein the first complementary region of the second oligonucleotide includes the 5' end of the second oligonucleotide. 17. The method according to any one of embodiments 1 to 16, wherein the third complementary region of the second oligonucleotide comprises the 3' end of the second oligonucleotide. 18. The method according to any one of embodiments 1 to 17, wherein the first complementary region of the second oligonucleotide is adjacent to the third complementary region of the second oligonucleotide. 19. The method according to any one of embodiments 1 to 18, wherein the barcode sequence of the second oligonucleotide provides barcode information for the identification of a target nucleic acid. 20. The method according to any one of embodiments 1 to 19, comprising contacting immobilized and permeabilized intact tissue to hybridize a plurality of oligonucleotide primers having specificity for different target nucleic acids. 21. The method according to embodiment 1, wherein the second oligonucleotide is provided as a closed nucleic acid ring, and the step of adding ligase is omitted. 22. Melting temperature of oligonucleotides (T m The method according to any one of embodiments 1 to 21, wherein the method is selected to minimize ligation in the solution. 23. The method according to any one of embodiments 1 to 22, wherein the addition of ligase includes the addition of DNA ligase. 24. The method according to any one of aspects 1 to 23, wherein the nucleic acid molecule comprises an amine-modified nucleotide. 25. The method according to aspect 24, wherein the amine-modified nucleotide comprises an N-hydroxysuccinimide moiety modification with acrylic acid. 26. The method according to any one of aspects 1 to 25, wherein embedding comprises copolymerizing one or more amplicons with acrylamide. 27. The method according to any one of aspects 1 to 26, wherein embedding comprises clarifying an amplicon embedded in one or more hydrogels, and the target nucleic acid is substantially retained in the amplicon embedded in one or more hydrogels. 28. The method according to aspect 27, wherein clarifying comprises substantially removing a plurality of cell components from an amplicon embedded in one or more hydrogels. 29. The method according to aspect 27 or 28, wherein clarifying comprises substantially removing lipids from an amplicon embedded in one or more hydrogels. 30. The method according to any one of aspects 1 to 29, wherein the third oligonucleotide is configured to decode bases. 31. The method according to any one of aspects 1 to 30, wherein the fourth oligonucleotide is configured to convert the decoded bases into signals. 32. The method according to aspect 31, wherein the signal is a fluorescence signal. 33. The method according to any one of aspects 1 to 32, wherein contacting an amplicon embedded in one or more hydrogels comprises eliminating the accumulation of errors as sequencing progresses. 34. The method according to any one of aspects 1 to 33, wherein imaging comprises imaging an amplicon embedded in one or more hydrogels using confocal microscopy, two-photon microscopy, brightfield microscopy, tissue-clearing expansion microscopy, and / or CLARITY (trademark) optimized light sheet microscopy (COLM). 35. The method according to any one of aspects 1 to 34, wherein the intact tissue is a thin section. 36. The method according to embodiment 35, wherein the intact tissue has a thickness of 5 to 20 μm. 37. The method according to embodiment 35 or 36, wherein the amplicons embedded in one or more hydrogels are brought into contact four or more times. 38. The method according to embodiment 35 or 36, wherein the amplicons embedded in one or more hydrogels are brought into contact five or more times. 39. The method according to any one of embodiments 1 to 34, wherein the intact tissue is a thick section. 40. The method according to embodiment 39, wherein the intact tissue has a thickness of 50 to 200 μm. 41. The method according to embodiment 39 or 40, wherein the amplicons embedded in one or more hydrogels are brought into contact six or more times. 42. The method according to embodiment 39 or 40, wherein the amplicons embedded in one or more hydrogels are brought into contact seven or more times. 43. A method for screening candidate drugs and determining whether the candidate drugs regulate the gene expression of nucleic acids in cells within intact tissue, (a) Contacting immobilized and permeabilized intact tissue with at least one pair of oligonucleotide primers under conditions that enable specific hybridization, The primer pair comprises a first oligonucleotide and a second oligonucleotide. Each of the first oligonucleotide and the second oligonucleotide comprises a first complementary region, a second complementary region, and a third complementary region, and the second oligonucleotide further comprises a barcode sequence. The first complementary region of the first oligonucleotide is complementary to the first portion of the target nucleic acid, the second complementary region of the first oligonucleotide is complementary to the first complementary region of the second oligonucleotide, the third complementary region of the first oligonucleotide is complementary to the third complementary region of the second oligonucleotide, the second complementary region of the second oligonucleotide is complementary to the second portion of the target nucleic acid, and the first complementary region of the first oligonucleotide is adjacent to the second complementary region of the second oligonucleotide, thus creating contact. (b) Adding ligase to ligate the second oligonucleotide and generate a closed nucleic acid ring, (c) Performing rolling circle amplification in the presence of nucleic acid molecules, comprising forming one or more amplicons using a second oligonucleotide as a template and a first oligonucleotide as a primer for polymerase, (d) Embedding one or more amplicons in the presence of hydrogel subunits to form amplicons embedded in one or more hydrogels, (e) Contacting an amplicon embedded in one or more hydrogels having a barcode sequence with a pair of primers under conditions that enable ligation, wherein the pair of primers comprises a third oligonucleotide and a fourth oligonucleotide, and ligation occurs only when both the third and fourth oligonucleotides ligate to the same amplicon. (f) Repeat step (e) many times, (g) Image amplicons embedded in one or more hydrogels to determine the in situ gene sequence of target nucleic acids in cells within intact tissue, (h) A method comprising detecting the level of gene expression of a target nucleic acid, wherein the change in the level of expression of the target nucleic acid in the presence of at least one candidate drug compared to the level of expression of the target nucleic acid in the absence of at least one candidate drug indicates that at least one candidate drug modulates gene expression of the nucleic acid in cells within intact tissue. 44. The method according to embodiment 43, wherein a pair of primers are denatured by heating before contact with the sample. 45. The method according to embodiment 43 or 44, wherein the cells are present within a population of cells. 46. ​​The method according to embodiment 45, wherein the cell population includes multiple cell types. 47. The method according to any one of embodiments 43 to 46, comprising contacting immobilized and permeabilized intact tissue to hybridize a pair of primers to the same target nucleic acid. 48. The method according to any one of embodiments 43 to 47, wherein the target nucleic acid is RNA. 49. The method according to embodiment 48, wherein RNA is mRNA. 50. The method according to any one of embodiments 43 to 47, wherein the target nucleic acid is DNA. 51. The method according to any one of embodiments 43 to 50, wherein the second oligonucleotide comprises a padlock probe. 52. The method according to any one of embodiments 43 to 51, wherein the first complementary region of the first oligonucleotide has a length of 19 to 25 nucleotides. 53. The method according to any one of embodiments 43 to 52, wherein the second complementary region of the first oligonucleotide has a length of 6 nucleotides. 54. The method according to any one of embodiments 43 to 53, wherein the third complementary region of the first oligonucleotide has a length of 6 nucleotides. 55. The method according to any one of embodiments 43 to 54, wherein the first complementary region of the second oligonucleotide has a length of 6 nucleotides. 56. The method according to any one of embodiments 43 to 55, wherein the second complementary region of the second oligonucleotide has a length of 19 to 25 nucleotides. 57. The method according to any one of embodiments 43 to 56, wherein the third complementary region of the second oligonucleotide has a length of six nucleotides. 58. The method according to any one of embodiments 43 to 57, wherein the first complementary region of the second oligonucleotide includes the 5' end of the second oligonucleotide. 59. The method according to any one of embodiments 43 to 58, wherein the third complementary region of the second oligonucleotide comprises the 3' end of the second oligonucleotide. 60. The method according to any one of embodiments 43 to 59, wherein the first complementary region of the second oligonucleotide is adjacent to the third complementary region of the second oligonucleotide. 61. The method according to any one of embodiments 43 to 60, wherein the barcode sequence of the second oligonucleotide provides barcode information for the identification of a target nucleic acid. 62. The method according to any one of embodiments 43 to 61, comprising contacting immobilized and permeabilized intact tissue to hybridize a plurality of oligonucleotide primers having specificity for different target nucleic acids. 63. The method according to embodiment 43, wherein the second oligonucleotide is provided as a closed nucleic acid ring, and the step of adding ligase is omitted. 64. Melting temperature of oligonucleotides (T m The method according to any one of embodiments 43 to 63, wherein the method is selected to minimize ligation in solution. 65. The method according to any one of embodiments 43 to 64, wherein the addition of ligase includes the addition of DNA ligase. 66. The method according to any one of embodiments 43 to 65, wherein the nucleic acid molecule comprises an amine-modified nucleotide. 67. The method according to embodiment 66, wherein the amine-modified nucleotide comprises a partial modification of N-hydroxysuccinimide acrylate. 68. The method according to any one of embodiments 43 to 67, wherein embedding comprises copolymerizing one or more amplicons with acrylamide. 69. The method according to any one of embodiments 43 to 68, wherein the embedding comprises clarifying amplicons embedded in one or more hydrogels, and the target nucleic acid is substantially retained within the amplicons embedded in one or more hydrogels. 70. The method according to embodiment 69, wherein clarification comprises substantially removing multiple cellular components from an amplicon embedded in one or more hydrogels. 71. The method according to embodiment 69 or 70, wherein clarification comprises substantially removing lipids from amplicons embedded in one or more hydrogels. 72. The method according to any one of embodiments 43 to 71, wherein the third oligonucleotide is configured to decode the base. 73. The method according to any one of embodiments 43 to 72, wherein a fourth oligonucleotide is configured to convert the decoded base into a signal. 74. The method according to embodiment 73, wherein the signal is a fluorescent signal. 75. The method according to any one of embodiments 43 to 74, wherein contacting amplicons embedded in one or more hydrogels eliminates the accumulation of errors as sequencing progresses. 76. The method according to any one of embodiments 43 to 75, wherein imaging comprises imaging an amplicon embedded in one or more hydrogels using confocal microscopy, two-photon microscopy, bright-field microscopy, intact tissue expansion microscopy, and / or CLARITY® optimized light sheet microscopy (COLM). 77. The method according to any one of embodiments 43 to 76, wherein the intact tissue is a thin section. 78. The method according to embodiment 77, wherein the intact tissue has a thickness of 5 to 20 μm. 79. The method according to embodiment 77 or 78, wherein the amplicons embedded in one or more hydrogels are brought into contact four or more times. 80. A method according to embodiment 77 or 78, wherein contacting an amplicon embedded in one or more hydrogels occurs five or more times. 81. A method according to any one of embodiments 43 to 76, wherein the intact tissue is a thick section. 82. A method according to embodiment 81, wherein the intact tissue has a thickness of 50 to 200 μm. 83. A method according to embodiment 81 or 82, wherein contacting an amplicon embedded in one or more hydrogels occurs six or more times. 84. A method according to embodiment 81 or 82, wherein contacting an amplicon embedded in one or more hydrogels occurs seven or more times. 85. A method according to any one of embodiments 43 to 84, wherein detecting comprises performing flow cytometry, sequencing, probe binding and electrochemical detection, pH change, catalysis induced by an enzyme bound to a DNA tag, quantum entanglement, Raman spectroscopy, terahertz wave technology, and / or scanning electron microscopy. 86. A method according to embodiment 85, wherein the flow cytometry is mass cytometry or fluorescence-activated flow cytometry. 87. A method according to any one of embodiments 43 to 86, wherein detecting comprises performing microscopy, scanning mass spectrometry, or other imaging techniques. 88. A method according to any one of embodiments 43 to 87, wherein detecting comprises determining a signal. 89. A method according to embodiment 88, wherein the signal is a fluorescence signal. 90. A system comprising: a fluid device including an imaging chamber and a pump; a processor unit configured to perform any one of embodiments 1 to 42. 【Examples】 【0115】 The following examples are provided to those skilled in the art to provide a complete disclosure and explanation of the methods of preparation and use of the present invention, and are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to represent that the following experiments are all or only experiments that can be performed. Although efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., quantity, temperature, etc.), some experimental error and deviation should be taken into account. Unless otherwise specified, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is in Celsius, and pressure is atmospheric pressure or near atmospheric pressure. Standard abbreviations may be used, such as bp (base pair); kb (kilobase); pl (picolitter); 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). 【0116】 material and method The following materials and methods generally apply to the results presented in the examples described herein, unless otherwise specified. 【0117】 mouse All animal procedures followed animal care guidelines approved by the Administrative Panel on Laboratory Animal Care (APLAC) at Stanford University and the National Institutes of Health guidelines. Male C57 / BL6 mice (6-10 weeks old) were used in the experiments. For dark / light experiments, mice were housed in a standard light cycle, followed by 5 days in constant darkness. After dark housing, mice were either sacrificed or exposed to light for 1 hour prior to sacrificial. For cocaine experiments, mice were injected with either saline or 15 mg / kg of cocaine 1 hour prior to sacrificial. For thin sections, animals were anesthetized with isoflurane and rapidly decapitated. Brain tissue was removed, placed in OCT, frozen in liquid nitrogen, and sliced ​​using a cryostat (Leica CM1900, see details in the thin tissue section below). For large sample volumes, animals were anesthetized with buprenex (100 mg / ml, ip), perfused transcardially with cold PBS, and then perfused with 4% PFA (see the section on large sample volumes below for further details). Thy1-YFP mice were B6.Cg-Tg(Thy1-YFP)HJrs / J. Transgenic pervalvumin mice were generated by mating Parv-IRES-Cre (JAX#8069) and Ai14 (JAX #7908) mice. 【0118】 Chemicals and enzymes The following chemicals and enzymes are listed by name (supplier, catalog number): Gel Slick solution (Lonza, 50640); PlusOne bind silane (GE Healthcare, 17-1330-01); Poly-L-lysine solution, 0.1 wt / vol% (Sigma, P8920); Ultrapure distilled water (Invitrogen, 10977-015); 12-well and 24-well glass-bottom plates (MatTek, P12G-1.5-14-F and P24G-1.5-13-F); #2 microcover slips, 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, 1x: 10010-023, 10x: 70011-044). 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). Ribonucleoside vanadyl 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 (Thermo Scientific, EP0094). 10 mM dNTP mixture (Invitrogen, 100004893). BSA, Molecular Biology Grade (New England) Biolabs, B9000S). 5-(3-aminoaryl)-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). Protease K, RNA grade (Invitrogen, 25530049). Alkali phosphatase (New England Biolabs, M0371L) DAPI (Molecular Probes, D1306). NeuroTrace fluorescent Nissl stain, yellow (Molecular Probes, N-21480). PMSF (Sigma, 93482). Papain (Worthington, LS003127). Matrigel (Corning Life Sciences, 356234). Neurobasal-A medium (Invitrogen, 21103-049). FBS (HyClone, SH3007103). B-27 supplement (Gibco, 17504044). 2 mM glutamax (Gibco, 35050-061). Fluorodeoxyuridine (Sigma, F-0503). Anti-NeuN antibody (Abcam, 190565). 【0119】 Primary mouse cortical neuron culture Neo-otis or hippocampal regions were removed from mouse offspring on postpartum day 0 (P0), digested with 0.4 mg / mL papain, and plated on 24-well glass-bottom plates pre-coated with 1:30 Matrigel at a cell density of 65,000 cells per well. Cultured neurons were maintained in Neurobasal-A medium containing 1.25% FBS, 4% B-27 supplement, 2 mM Glutamax, and 2 mg / mL fluorodeoxyuridine, and kept in a moist culture incubator with 5% CO2 at 37°C. 【0120】 smFISH Stellaris ShipReady smFISH probes for mouse Gapdh containing Quasar570 were purchased from LGC Bioresearch Technologies (SMF-3002-1). smFISH experiments were performed according to the manufacturer's protocols for adherent cells and frozen mouse brain tissue. 【0121】 STARmap probe design The SNAIL probes were designed as follows: (1) The consensus cDNA sequences (CCDS) of all mouse proteins were downloaded from ftp: / / ftp.ncbi.nlm.nih.gov / pub / CCDS / current_mouse, and the reference transcriptome was downloaded (hgdownload.cse.ucsc.edu / goldenPath / mm10 / bigZips / ). For genes with multiple transcription isoforms, only the shortest isoform was considered. (2) Using Picky2.2 (32-bit), hybridization sequences for each probe pair were designed with a length limit of 40-46 nucleotides, designing four sequences for each gene. (3) The resulting complementary DNA (cDNA) sequences (40-46 nt) were split into 20-25 nt halves, which had a 0-2 nt gap between them and had the best match in melting temperature (Tm) between the two halves. All probes were less than 60nt and were manufactured as 96-well plates by Integrated DNA Technologies (IDT). For experiments with 160 transcripts, the homemade sequencing reagent included six read probes (R1-R6) labeled with Alexa 488, 546, 594, and 647, as well as 16 2-nucleotide encoded fluorescent probes (2nucleotide_F1-2nucleotide_F16). For large-scale experiments with 28 transcripts, all SNAIL primer probes were ordered with acridite modification, and sequencing was performed using 11nt orthogonal read probes (OR1-OR7) and four single-nucleotide fluorescent probes (1nucleotide_F1-1nucleotide_F4). All sequences are available as Excel data files on the publisher's website. 【0122】 STARmap procedures for cell cultures and thin tissue sections Sample preparation Twelve-well or twenty-four-well glass-bottom plates were treated with methacrylateoxypropyltrimethoxysilane (bindsilane). For brain tissue sections, twelve-well plates were further treated with poly-L-lysine solution. #2 microcover slips (12 mm) were pre-treated with Gel Slick for subsequent polymerization according to the manufacturer's instructions. Primary neuron cell cultures were fixed in 1.6% PFA in PBS for 10 minutes, then transferred to pre-cooled (-20°C) methanol and maintained at -80°C for at least 15 minutes (and up to one week). For brain tissue, freshly harvested mouse brains were immediately embedded in OCT and snap-frozen. The tissue was either stored at -80°C or transferred to a cryostat and cut into 16 μm sections. Sections containing the primary visual cortex were mounted on pre-treated glass-bottom plates. Brain sections were fixed in 4% PFA in PBS at room temperature for 10 minutes, permeabilized with methanol at -20°C, then left at -80°C for 15 minutes before hybridization. 【0123】 Library construction SNAIL probes were dissolved in ultra-high purity RNase-free water at 100 or 200 μM and pooled. The probe mixtures were heated at 90°C for 2–5 minutes and then cooled to room temperature. Samples were taken from -80°C, equilibrated at room temperature for 5 minutes, washed with PBSTR (0.1% Tween-20, 0.1 U / μL SUPERase·In in PBS) for 2–5 minutes, and incubated overnight in a humidified oven at 40°C with gentle shaking in 1× hybridization buffer (2× SSC, 10% formamide, 1% Tween-20, 20 mM RVC, 0.1 mg / ml salmon sperm DNA, and pooled SNAIL probe at 100 nM per oligo). The samples were then washed twice with PBSTR for 20 minutes each, followed by a single 20-minute wash in 4× SSC dissolved in PBSTR at 37°C. Finally, the sample was briefly rinsed once with PBSTR at room temperature. The sample was then incubated for 2 hours at room temperature with a T4 DNA ligation mixture (1:50 dilution of T4 DNA ligase supplemented with 1×BSA and 0.2 U / μL of SUPERase-In) with gentle stirring. The sample was then washed twice with PBSTR and incubated with an RCA mixture (1:50 dilution of Phi29 DNA polymerase, 250 μM dNTP, 1×BSA and 0.2 U / μL of SUPERase-In, and 20 μM of 5-(3-aminoaryl)-dUTP) with stirring at 30°C for 2 hours. The sample was then washed twice in PBST (PBSTR without SUPERase-In) and treated with 20 mM NHS acrylate in PBST at room temperature for 2 hours. After a brief wash with PBST, the sample was incubated with monomer buffer (4% acrylamide, 0.2% bisacrylamide, 2×SSC) at room temperature for 30 minutes. The buffer was aspirated, and 10 μL of polymerization mixture (0.2% ammonium persulfate, 0.2% tetramethylenediamine dissolved in monomer buffer) was added to the center of the sample. These were immediately covered with a coverslip coated with Gel Slick, incubated at room temperature for 1 hour, and then washed twice with PBST for 5 minutes each time.The tissue gel hybrid was digested with proteinase K (0.2 mg / ml) at 37°C for 1 hour, and then washed three times with PBST (5 minutes each time). 【0124】 Imaging and sequencing For single-gene detection, a 19nt fluorescent oligo complementary to the DNA amplicon was diluted to 500 nM in 1× SSC dissolved in PBST, and the sample was incubated at room temperature for 30 minutes, then washed three times with PBST for 5 minutes each, before imaging. For sequencing, each sequencing cycle began with treating the sample twice with stripping buffer (60% formamide, 0.1% Triton-X-100) for 10 minutes at room temperature, followed by three PBST washes of 5 minutes each. The sample was incubated with the sequencing mixture (1× T4 DNA ligase buffer, 1:25 dilution of T4 DNA ligase, 1× BSA, 10 μM read probe, and 5 μM fluorescent oligo) at room temperature for 3 hours. The sample was then rinsed three times each (10 minutes each) with washing and imaging buffer (2× SSC and 10% formamide) before proceeding to imaging. DAPI staining was performed before cycle 1 and after cycle 6, according to the manufacturer's instructions, for the purpose of aligning sequencing images with Nissl staining. Nissl staining was performed after cycle 6, according to the manufacturer's instructions, for the purpose of cell segmentation. Images with a voxel size of 78 nm × 78 nm × 315 nm were acquired using a Leica TCS SP8 confocal microscope equipped with a 405 diode, white light laser, and a 40x oil immersion objective lens (NA 1.3). 【0125】 Thin section STARmap data processing All image processing steps were performed using MATLAB® R2017A. 【0126】 Image alignment Image alignment was achieved using a three-dimensional Fast Fourier Transform (FFT) to calculate the cross-correlation between two image volumes at all translational offsets. The location of the highest correlation coefficient was identified and used to translate the image volume and compensate for the offset. All images were aligned for the first round of sequencing, first through a global transformation across the entire field of view, and then separately for each tile (corresponding to the individual tiled fields of view used by the microscope during image acquisition). 【0127】 Spot Call After alignment, individual dots were identified separately in each color channel during the first round of sequencing. For 160 gene experiments, dots with a diameter of approximately 7 pixels were identified by first filtering the volume with a 3D Laplacian of Gaussian, and then finding the local maximum in 3D. For 1020 gene experiments, dots were found using a similar method, but the Laplacian of Gaussian was calculated at multiple scales, and the maximum was found across these scales. After identifying each dot, the dominant color of the dot across all four channels was determined in each round by the 3×3×1 voxel volume surrounding the dot location. 【0128】 Barcode filtering The dots were first filtered based on a quality score. The quality score quantified the range obtained from a single color rather than a mixture of colors for each dot in each sequencing round. The barcode codebook was converted to a color space based on the expected color sequence after 2-base encoding of the barcode DNA sequence. The color sequences of dots that passed the quality threshold and matched sequences in the codebook were retained, identified by the specific gene represented by that barcode, and all other dots were rejected. High-quality dots and their associated genetic identities in the codebook were then saved for downstream analysis. 【0129】 2D cell segmentation Nuclei were manually identified from the maximum intensity projection of DAPI channels after the final round of sequencing. Cell bodies were initially identified using a random forest classifier run in Ilastik based on Nissl staining. The classifier was trained on a randomly selected subset of trimmed regions from all samples and then applied to the entire image. The thresholded probability map was then pixel-scaled to fill in the remaining gaps. Finally, a marker-based watershed transformation was applied to segment the combined, thresholded cell body map and the thresholded cell bodies based on the identified nuclei locations. A convex hull was calculated around each cell body. The gene expression matrix per cell was then calculated by assigning points overlapping each convex hull in 2D to that cell. 【0130】 Single-cell data preprocessing All single-cell analyses were performed using a custom Python software package for analyzing STARmap experiments. The expression matrix per cell was first normalized for the expression values ​​Eij across all genes j for each cell i using the following formula. 【0131】 N ij =ln(1+median(E) i: ) * (E ij / ΣE i: )) 【0132】 For clustering, we used a linear model to regression the effects related to the number of transcripts per cell, sample identity, and experimental conditions (light vs. dark). 【0133】 E ij =nSpots i +exptID i +exptCond i And E ij It is assumed that the distribution follows a Poisson distribution. Top-level single-cell clustering 【0134】 After normalization and scaling, principal component analysis was applied to reduce the dimensionality of the cell expression matrix. Then, based on the explained variance ratios, the top PCs were used for top-level clustering, based on a manual analysis of the explained variance ratios per PC. Next, the top PCs were clustered using a shared nearest neighbor (SNN) algorithm with Leuven distance. Clusters enhanced for the excitatory neuronal marker Slc17a7 (vesicular glutamate transporter), the inhibitory neuronal marker Gad1, and the non-neuronal marker MQp were manually integrated to form three major clusters representing these cell types. Cells were visualized using Uniform Manifold Approximation and Projection (UMAP) (https: / / github.com / lmcinnes / umap). Then, the cells in each cluster were subclustered using PCA decomposition, followed by Leuven SNN clustering to determine specific cell types. 【0135】 Subclustering of single cells Next, the inhibitory, excitatory, and non-neuronal clusters were subclustered using the same approach applied to the primary clusters. 【0136】 Differential expression analysis Genes that were specifically variable within this subcluster were selected by calculating the P-values ​​of differential expression for each gene between each cluster and all other clusters using the Bimod test (likelihood ratio test). The P-values ​​were FDR-corrected based on the number of clusters. 【0137】 STARmap procedure for thick tissue sections Sample preparation Twelve-well glass-bottom plates and microcover slips (12 mm) were pre-treated in the same manner as for thin tissue sections. Mice were perfused with PFA, fixed on ice for 2-3 hours, transferred to PBS on ice for 30 minutes, and cut into 150 μm sections. Sections containing the primary visual cortex were transferred to the pre-treated glass-bottom plates and washed once with ice-cold PBS. 【0138】 Library construction The samples were pre-clarified with methanol cooled to -20°C at 4°C for 1 hour, and then incubated in 1× permeabilization and hybridization buffer (2× SSC, 10% formamide, 1% Triton-X-100, 20 mM RVC, 0.1 mg / ml salmon sperm DNA and pooled SNAIL probe at 100 nM per oligo, and 0.2% SDS) in a humidified oven at 40°C for 2 days with gentle shaking. The methanol treatment was skipped to protect the samples from swelling and deformation and to allow for co-detection of protein and RNA (Thy1-YFP). The samples were then washed twice with PBSTV (0.1% Triton and 2 mM RVC) at 37°C for 1 hour each, followed by a further wash with PBS for 1 hour. Next, the sample was incubated with polymerization mixture I (4% acrylamide, 0.2% bis-acrylamide, 2×SSC, 0.5% VA-044) at 4°C for 1 hour. The buffer was aspirated, and 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. These were immediately covered with a coverslip coated with Gel Slick and incubated at 40°C for 1 hour. The sample was then 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 in 2×SSC, 1% SDS), and then washed twice with PBSF (2 mM PMSF in PBS) and PBSTR (30 minutes each time). Next, the samples were incubated with a T4 DNA ligation mixture (1:50 dilution of T4 DNA ligase supplemented with 1× BSA and 0.2 U / μL SUPERase-In) at room temperature for 12 hours with gentle stirring. Then, the samples were washed twice with PBSTR (1 hour each time), and then incubated with an RCA mixture (1:50 dilution of Phi29 DNA polymerase, 250 μM dNTPs, 1× BSA and 0.2 U / μL SUPERase-In, and 20 μM 5-(3-aminoaryl)-dUTP) at 30°C for 12–24 hours. Finally, the samples were crosslinked with BSPEG9. 【0139】 Imaging and sequencing First, the sample was stained overnight with DAPI, then rinsed with PBS for 1 hour. Each cycle began with treating the sample twice with stripping buffer (60% formamide, 0.1% Triton-X-100) for 20 minutes at room temperature, followed by three PBST washes of 20 minutes each. The sample was incubated with a large sequencing mixture (1× T4 DNA ligase buffer, 1:50 dilution of T4 DNA ligase, 1× BSA, 5 μM orthogonal reading probe, and 400 nM single-nucleotide fluorescent oligo) at room temperature for 4 hours. The sample was then rinsed twice with PBS (20 minutes each) before proceeding to imaging. Leica TCS equipped with a 405 nm diode, white light laser, and 25x water immersion objective lens (NA 0.95) Using an SP8 confocal microscope, we acquired images with a voxel size of 0.9 μm × 0.9 μm × 1 μm. 【0140】 Processing large amounts of STARmap data The image registration 3D FFT alignment was applied again, except that DAPI channels were used for alignment. Specifically, the DAPI channels in each round were globally aligned in the first round, and then piecewise aligned within a 4x5 grid corresponding to the field of view tiles used to acquire the images. 【0141】 Cellular findings and quantification After alignment, cells were identified using the minimum value of the Laplacian-of-Gaussian filter applied to the DAPI channel. To quantify the expression of each gene, the average intensity in each color channel was averaged over a 10 × 10 × 3 voxel volume around each nucleus. 【0142】 3D cell analysis First, cells were clustered into inhibitory, excitatory, and non-neuronal types using k-means, employing Gad1, Slc17a7, and several non-neuronal genes. Then, each cluster was subclustered using k-means. The initial values ​​for k-means were set to the average expression of each marker gene. To calculate the distances between cell types, the nearest neighbor distances between all excitatory neurons and each inhibitory neuron subtype were calculated using a kD tree for fast nearest neighbor calculations with Euclidean distances. 【0143】 Actb spike-in to evaluate the physical limits of STARmap All 100 and 1000 genes shared a common 18nt sequence (sequence A) in the padlock that would be amplified in the final nanoball. A separate set of SNAIL probes was designed for Actb, which has a different 18nt sequence (sequence B). The SNAIL probes for Actb and the SNAIL probes for the 0, 100, or 1000 genes were mixed together and used for hybridization. Both the Actb spike-in and the 100 and 1000 genes underwent the same ligation and amplification steps to ensure equal efficiency. For readout, two fluorescent detection oligos (Alexa488-probe complementary to sequence B and Alexa647-probe targeting sequence A) were added to the sample, and the amplicons of Actb and the remaining genes were imaged in two separate channels. Next, we tested the amplicons of Actb RNA to determine whether the number of amplicons was diluted by the increased number of other genes, using this as an indicator of molecular crowding (Figures 16A-16E). 【0144】 CLARITY tissue immunohistochemical staining PFA-fixed tissues were processed as described in Shendure et al (2005). Briefly, 200 μm thick PFA-fixed brain sections were placed in a 1% acrylamide embedding solution at 4°C for 23 hours, then embedded at 37°C for 4 hours, and subsequently clarified passively for 5 days. The clarified sections were washed in PBST for 2 days, stained with anti-NeuN (1:100) at room temperature for 24 hours, and then washed in PBST for 24 hours. The sections were imaged using a confocal microscope. 【0145】 A device for automated in-situ sequencing (hypothesis) The automated steps of the experimental procedure include three buffers, maintained at room temperature in larger volumes: 1) wash buffer, 2) imaging buffer, and 3) strip buffer. Tubes containing the ligase enzyme mixture are maintained at 4°C. There are eight tubes containing fluorescently labeled imaging material, maintained at 4°C. Six of the tubes contain fluorescently labeled oligonucleotides and probes for hybridization. One of the tubes contains small molecule Nissl stain, and one contains small molecule DAPI stain. If it is impossible to mix the ligase and oligos at imaging, they can potentially be pre-mixed and maintained at 4°C. 【0146】 The sequencing process involves a custom imaging chamber containing the sample connected to a fluid. For each of the six rounds, a mixture of ligase, ligase, and one of the six oligonucleotide mixtures (total volume 200-400 μl) is applied to the sample for 3 hours. Next, wash buffer is run over the sample for 10 minutes. Then, the wash buffer is replaced with imaging buffer. The system then sends a trigger to the microscope, which performs fluorescence imaging. Once imaging is complete, the microscope sends a trigger to the system to begin the next round. The system runs stripping buffer over the sample for 10 minutes. Wash buffer is run for 5 minutes to remove any remaining stripping buffer. The ligase and fluorescently labeled oligo for the next round are applied. After repeating this process six times, the laser rounds are stripped, washed, and then Nissl and DAPI solutions are applied simultaneously for 1 hour. This is then washed and imaged with imaging buffer. Finally, after the experiment is complete, the entire system is rinsed with bleach to clean it for the next run. 【0147】 The computer-controlled fluid pumps various solutions, mixes ligase and oligo solutions, and selects the oligo solution to be used in each round. All parameters in this sequence (flow rate, time, etc.) should be programmable. 【0148】 For all this, a custom imaging chamber holds small-volume, sealed tissue sections with inlet and outlet ports and fits into a microscope equipped with a 24-plate holder. Figure 24A depicts the overall hardware setup for STARmap sequencing, and Figure 24B depicts the design of the exemplary fluid system described herein. 【0149】 result Example 1: STARmap The methods disclosed herein, including in-situ sequencing techniques, enable highly multiplexed gene detection (up to 1000 genes) in intact biological tissue. Starting from raw biological tissue (fresh or preserved, as small as a single cell or at most 1 millimeter in size), these methods result in gene quantification based on output images with subcellular and cellular resolution, high efficiency, low error rates, and fast processing times. 【0150】 A schematic diagram of the STARmap as described herein is shown in Figure 1A. After preparing brain tissue (see Methods for the mouse brain protocol), a custom SNAIL probe (Figure 2A) was encountered in intact tissue and hybridized with intracellular mRNA (dashed line), and then enzymatically replicated as a cDNA amplicon. The amplicon was constructed in situ using partial modification of N-hydroxysuccinimide acrylate, then copolymerized with acrylamide to embed in a hydrogel network (wavy line), and subsequently, Unbound lipids and proteins were then cleared (Figures 3A–3F). Each SNAIL probe contained a gene-specific identifier segment, which was read out via in-situ sequencing using 2-nucleotide encoding for error correction (SEDAL, Figures 4A–4L). Finally, highly multiplexed RNA quantification in 3D revealed gene expression and cell type in space. 【0151】 Figure 1A depicts the following method for SNAIL, where primer-padlock probe pairs amplified a target-specific signal, eliminating noise known to commonly arise from nonspecific hybridization of single probes. Figures 1C and 1D show that only adjacent joining of primer-padlock probes resulted in signal amplification. mRNA A represents Gapdh and mRNA B represents Actb. Both fluorescence images show Gapdh mRNA (gray) and nucleus (blue) labeling in mouse brain sections, highlighting the absence of labeling by mismatched primers and padlocks (Figure 1D, right). The scale bar indicates 10 μm. Figure 1E depicts in-situ sequencing of DNA amplicons in tissue-hydrogel complexes via SEDAL, an improved ligation sequencing method devised for STARmap. For each cycle, the reading probe (lines without star symbols) contained a progressively increasing length run of degenerate bases (N representing an equal mixture of A, T, C, and G) with a phosphate (5'P) at the 5' end to establish the reading position, while the decoding probe (lines with star symbols) was labeled with a fluorophore having a color coding for dinucleotides at the 3' end. The two probes were ligated only if both probes were perfectly complementary to the DNA template (lower sequence) to form a stable product with a high melting temperature, allowing for subsequent imaging after washing away the unligated probe. After each imaging cycle, the probes were exfoliated from the robust tissue-hydrogel using 60% formamide so that the next cycle could be started. X indicates the unknown base to be read, "underlined" indicates the decoded sequence, and Ch1-4 indicates the fluorescence channel. The scale bar represents 2 μm. 【0152】 Example 2: SNAIL Reverse transcription can be a major efficiency limiting step for in-situ sequencing, and SNAIL bypassed this step with a primer-padlock probe pair (Figure 2A) designed only when both probes hybridize to the same RNA molecule. The design of the SNAIL probe (one component of STARmap) includes the following, and each primer or padlock probe (19-25 nucleotides; nt, blue) is designed to hybridize with the target RNA at temperatures above 60°C. m (It had a nucleic acid melting temperature), but the complementary sequence between the primer and padlock was T m However, since the amount of DNA-DNA hybridization of the primer-padlock was only 6nt on each arm at below room temperature, it was minimal during DNA-RNA hybridization at 40°C, but enabled DNA ligation by T4 DNA ligase in the next step. 【0153】 Padlock probes were cyclized, and rolling circles were amplified to generate cDNA nanoballs (amplicons) containing multiple copies of cDNA (Figures 1A-1D). This mechanism ensured target-specific signal amplification and eliminated noise that would otherwise inevitably arise from non-specific hybridization of single probes. Indeed, the outcomes were significantly higher absolute intensity and signal-to-noise ratio (SNR) compared to commercially available single-molecule fluorescence in situ hybridization (smFISH) probes (Figures 2B-2F). Figure 2B depicts a comparison of signal-to-noise ratio (SNR; mean intensity of signal spot / mean intensity of background) of commercially available smFISH probes and SNAIL probes targeting Gapdh mRNA in mouse cortical cell cultures and mouse visual cortex sections. Error bars indicate the standard deviation (sd) of the spot intensity. Error bars represent the standard deviations (sd) of 39,398, 30,297, 97,555, and 19,392 pixels, corresponding to RNA signals in 640,000 pixels of the acquired image, with *** indicating P<0.0001 by Student's t-test. Figures 2C-2F depict fluorescence images of Gapdh smFISH (Figures 2C and 2E) and SNAIL probe (Figures 2D and 2F) in cortical cell cultures (Figures 2C and 2D) and visual cortical sections (Figures 2E and 2F), with scale bars indicating 10 μm. Figure 2G shows a comparison of multiplexed RNA imaging methods using rolling circle amplification (RCA). Compared to FISSEQ and padlock probes, the SNAIL probe overcomes the efficiency limiting step of reverse transcription, significantly simplifying the experimental procedure. While PLAYR requires four probes, one additional step, and two ligation sites, SNAIL requires only one pair of probes and one ligation site. Box plots of RNA per cell for 151 cell type gene markers were measured by single-cell RNA sequencing (scRNA-seq, .ref) and STARmap (extracted from 160 gene mappings of the visual cortex) (Figure 2G).The box plots depict the first and third quartiles, the median as the center line, the whiskers as the 5% and 95% data points, and the p-values ​​as rank-sum tests. Summaries and numbers of single-cell RNA sequencing and RCA-based multiplexed RNA detection methods were extracted from the reference literature (Figure 2I). 【0154】 Example 3: Embedding cDNA amplicons into tissue-hydrogel settings To enable cDNA amplicon embedding into tissue-hydrogel settings, amine-modified nucleotides were spiked into a rolling circle amplification reaction, functionalized at the acrylamide moiety using N-hydroxysuccinimide acrylate, and copolymerized with the acrylamide monomer to form a hydrogel (Figure 1A, Figure 3A). A schematic diagram of the hydrogel histochemistry for STARmap in thin tissue sections is depicted in Figure 3A. DNA amplicons were synthesized in the presence of minute levels of 5-(3-aminoaryl)-dUTP, which slowly replaced T, so that the DNA amplicons were covalently immobilized at numerous sites within the polyacrylamide network, and further functionalization at the polymerizable acrylamide moiety was enabled using N-hydroxysuccinimide acrylate (AA-NHS) (Figure 3A, right). The resulting tissue-hydrogels were then subjected to protein digestion and lipid desorption to enhance clarity (Figures 3B-3E). Fluorescence images (total intensity from all four fluorescence channels) represent the detection of 160 genes in the mouse visual cortex (Figures 3B and 3C) and 16 genes in the mouse medial habenula (Figures 3D and 3E). Compared to untreated samples (Figures 3B and 3D), samples treated with 5-(3-aminoaryl)-dUTP H TC for lipid and protein clarification (Figures 3C and 3E) showed reduced opacity and autofluorescence. The scale bar represents 50 μm. DNA-gel crosslinking maintained DNA amplicons within the gel. Samples of 160 genes were prepared with or without AA-NHS and imaged in the medial prefrontal cortex (Figures 3F-3G). Fresh samples prepared without AA-NHS showed a 36% signal loss compared to AA-NHS-treated samples, and underwent a further 40% signal loss after storage at room temperature for 24 hours, while AA-NHS-treated samples showed only a 9% signal change (Figure 3F). Fluorescence intensity was the average of four technical replicas with imaging dimensions of 120 μm × 120 μm × 3 μm. Error bars indicate mean ± sd, and *** indicates p < 0.001 in a two-sided t-test. Fluorescence images are depicted in Figure 3G with a 3.5 μm scale bar. 【0155】 This design chemistry determined that the amplicon was covalently bound to the hydrogel network, thus maintaining the target's location and shape throughout multiple detection cycles. Figure 3H depicts zoom-in fluorescence images of a single neuron in the visual cortex detecting Gapdh RNA by STARmap for cycle 1, exfoliation, and cycle 2, as well as an integrated image of cycles 1 and 2, demonstrating the stable spatial position of the DNA amplicon across sequencing cycles. The scale bar represents 2 μm. 【0156】 Example 4: SEDAL As gene-specific identifiers to be sequenced, a 5-base barcode (library size of 1,024) was designed and incorporated into each padlock probe, thus enabling multiplexed gene detection (Figure 1A). The synthesis-based sequencing paradigm required an increased reaction temperature compared to ligation-based sequencing methods performed at room temperature, which was avoided because it posed problems for imaging and sample stability. However, none of the reported or commercially available ligation-based sequencing methods achieved the necessary SNR or accuracy for this challenging intact tissue application (Figures 4A-4L). For this reason, a novel method called SEDAL was devised, specifically for STARmap (Figures 4A-4L). 【0157】 A schematic diagram of SEDAL is shown in Figure 4A. SEDAL includes a T4 DNA ligase with activity strongly inhibited by base mismatch, and two types of sequencing probes: a reading probe that sets the base position to be investigated, and a fluorescence decoding probe that converts the base information into color for imaging. Unlike other ligation sequencing methods that use a pre-annealed reading probe (or equivalent), the reading probe in SEDAL is short (11nt, close to room temperature). mThe read probe was partially degenerate (for example, as shown in Figure 4A, cycle 4, the first two bases at the 5' end are N and the mixture is an equal amount of A, T, C, and G) and mixed with the decoding probe and T4 DNA ligase for a one-step reaction. At room temperature, the read probe remained in a dynamic state, annealing with the DNA template and detaching from the DNA template. The T4 DNA ligase ligated the read probe to the 8nt fluorescent decoding probe only when the read probe had a perfect match with the DNA template. That is, the two probes were ligated to form a stable product for imaging only when a perfect match occurred. The short read and decoding probes were then washed away, leaving the 19nt fluorescent product stably hybridized to the DNA amplicon for imaging. For the next cycle, the previous fluorescent product was detached, and the read probe contained one or more degenerate bases to shift the read frame by one base fraction (Figure 1E). 5'P indicated the 5' phosphate. 3'InvT indicates a 3' inverted dT base to prevent self-ligation of the read probe, and 3'OH indicates a 3' hydroxyl group. 【0158】 After each cycle corresponding to base readout, the fluorescent product was detached with formamide, thereby eliminating the accumulation of errors as sequencing progressed (Figures 1E and 4B). A comparison of the key characteristics of all ligation sequencing methods is shown in Figures 4C–4F. Background issues associated with commercially available SOLiD sequencing kits when applied to mouse brain tissue are depicted in Figures 4C and 4D, while the custom SEDAL reagent showed minimal background in Figures 4E and 4F. Signal images (Figures 4C and 4E) represent the first cycle of sequencing for the Malat1, Actb, Calm1, and Snap25 genes. Background images (Figures 4D and 4F) were acquired after cleavage / detachment of the first cycle. The scale bar represents 50 μm. 【0159】 Schematic diagrams of the single-base and double-base encoding paradigms are depicted in Figures 4G and 4H, along with exemplary results with or without a single sequencing error (incorrect color) during cycle 3. For single-base encoding, a single sequencing error resulted in a single base mutation, and therefore an incorrect 5-base code (Figure 4G). For double-base encoding, the 6-cycle paradigm played a role in error reduction. Incorrect readings were rejected because a single error propagated during any sequencing cycle, mutating an adjacent known base G to another base (Figure 4H). The double-base encoding scheme was designed and implemented to mitigate any residual errors associated with imaging high spot density (Figures 4G-4H). 【0160】 Based on a panel of four highly expressed test genes, the error rate of STARmap was more than an order of magnitude lower than previous methods (approximately 1.8% vs. 29.4%, Figures 4I-4L). Actual data from cPAL (representing a single-nucleotide encoding scheme) and SEDAL (representing a double-nucleotide encoding scheme) were applied to the detection of four genes in the mouse visual cortex. The SNAIL probes for Malat1, Actb, Calm1, and Snap25 were identical under both conditions, and the Hamming distance of each pair of the four 5-nucleotide codes was 5 (i.e., complete non-homologousity). Using such sparse coding, the sequencing error rate was estimated by the percentage of incorrect spots (not the four 5-nucleotide codes used) out of all detected spots (Figures 4I-4J). The spatial maps of the four genes detected by cPAL are shown in Figure 4I, and SEDAL is shown in Figure 4J. The error rate of cPAL in four gene experiments (Figure 4I) was 29.4% (Figure 4K), while the error rate of SEDAL after built-in error reduction (Figure 4J) was 1.8% (Figure 4L). 【0161】 Example 5: Cell classification of cell types in the primary visual cortex using STARmap To test whether STARmap could deliver the first target for high-content 3D intact tissue sequencing of single-cell transcriptional states with the required sensitivity and accuracy, we applied STARmap to the urgent current challenge in neuroscience: the detection and classification of cell types in the neocortex of the adult mouse brain, as well as the principles of corresponding tissue composition. While the biostructure and function of the mouse primary visual neocortex have been extensively studied, allowing for validation of results by comparing with previous findings across numerous papers, methodologies, and data sources, the full diversity of deeply molecularly defined cell types within the visual cortex remains spatially unresolved in a single experiment, potentially excluding the identification of fundamental collaborative statistics and tissue principles across 3D volume. Among the many examples of experimental impacts such information could provide, collaborative 3D cell typology mapping was used to aid in deciphering the spatiotemporal logic of neuronal activity-induced gene expression as a function of cell type and spatial location. 【0162】 The primary visual cortex (V1) was coronally sectioned, and a 5-nucleotide barcoded SNAIL probe was used over six rounds of in-situ SEDAL sequencing in coronally mouse brain sections (Figure 1A, Figures 5A–5B) to investigate a large, hardened gene set of 160 genes, including 112 putative cell type markers matched from mouse cortical single-cell RNA sequencing, and 48 activity-regulated genes (ARGs). In one arm of the mouse cohort, visually induced neuronal activity was provided via 1 hour of light exposure after 4 days of containment in the dark, while other mice were continuously kept in the dark before sacrifice. Volumes of 8 μm thickness containing 600–800 cells covering all cortical layers were imaged. Figure 5B depicts raw fluorescence images of STARmap during the process, along with a full image of cycle 1 (Figure 5B, top) and zoom views over all six cycles (Figure 5B, bottom). The full field of view is 1.4 mm × 0.3 mm, the scale bar is 100 μm, the zoomed area is 11.78 μm × 11.78 μm, the scale bar is 2 μm, the channels are color codes for four fluorescent channels, L1-6 represent six neocortical layers, cc represents the corpus callosum, and HPC represents the hippocampus. 【0163】 After six rounds of sequencing, Nissl fluorescence staining was used to segment the cell bodies and allow for the assignment of amplicons to individual cells (Figure 6A-6B). Figure 6A depicts a diagram illustrating the processing pipeline for extracting readouts decoded from raw imaging data (see also the Methods for details corresponding to each step), (1) imaging the sample over multiple rounds, (2) aligning the sample over the rounds, showing two rounds (green and purple) with inconsistencies that must be corrected by the alignment, (3) automatically identifying spots in each color channel as estimated amplicons that would be decoded based on the color values ​​at points in each round (independently in the first round), (4) recalling readouts based on a comparison of the maximum intensity of each spot over each round with the predicted color sequence for each barcoded DNA sequence (color space encoded barcode), (5) segmenting Nissl-containing cells from the background by detecting cells using machine learning-based segmentation that takes into account various intensity and texture features (described in Figure 6B), and (6) assigning readouts to cells by calculating the overlap between the location of each valid readout and the convex hull of the segmented region of each cell. 【0164】 Genes encoded by the cell's convex hull and overlapping readouts were assigned to that cell. Figure 6B illustrates the method for determining cell extents: (1) a random forest classifier (a non-parametric machine learning algorithm for marker prediction) was trained on a subsampled set of Nissl-stained data to distinguish cell-containing regions from the background; (2) cell locations were manually selected using DAPI (nuclear) channels; (3) the classifier was applied to the entire image to predict cell locations; and (4) cells were segmented from this prediction using marker-based watersheds, which segmented the cell-labeled regions of the image into distinct cell bodies based on the known locations of the nuclei. 【0165】 The histograms in Figure 5C detected reads (DNA amplicons) per cell (Figure 5C, left) and genes per cell (Figure 5C, right). While the values ​​corresponding to amplicons and genes per cell varied significantly (Figure 5C), the expression patterns of the 160 genes remained consistent across biological replications (R=0.94–0.95, Figure 5D), demonstrating reliable detection of transcript diversity at the single-cell level. Quantitative reproducibility of biological replication, regardless of light or dark conditions, is shown in Figure 5D, with log2 (amplicon amount) relating to the 160 genes across the entire plotted imaging area. rep1 represents the formula value in the first replication, and rep2 represents the formula value in the second replication. 【0166】 These 160 gene pilots faithfully reproduced known cortical layer markers and the spatial distribution of interneurons, as illustrated here through a comparison of in-situ images from paired public mapping projects with STARmap results (Figure 5E). The validation of STARmap shown in Figure 5E depicts in-situ images from the Allen Institute of Brain Science (AIBS) (Figure 5E, left column) and RNA patterns of individual genes extracted from 160 gene STARmaps that reliably reproduced spatial gene expression patterns from AIBS (Figure 5E, right column). 【0167】 Cell classification was performed using expression data for 112 cell type markers. First, more than 3,000 cells pooled from four biological replicas were clustered into three major cell types (excitatory neurons, inhibitory neurons, and non-neuronal cells) using graph-based clustering after principal component decomposition, and then further subclustered under each category (Figures 5F-5H and 6C). Uniform manifold approximation (UMAP) plots, a nonlinear dimensionality reduction technique used to visualize the similarity of cell transcriptomes in two dimensions, showed consistent clustering of major cell types across 3,142 cells, 2,199 excitatory neurons, 324 inhibitory neurons, and 619 non-neuronal cells pooled from four biological replicas (Figure 5F). Gene expression heatmaps for the 112 cell type markers shown in Figure 5G are consistent with each cell cluster and show clustering by inhibitory, excitatory, or non-neuronal cell type. The expression of each gene was z-scored across all genes within each cell. Representative cyto-degraded spatial maps of the neocortex and beyond are shown in Figure 5H, and cell types are coded as in Figure 5F. Clustering of excitatory and inhibitory subtypes (Figures 5I-5N) is depicted with UMAP plots (Figures 5I and 5L), bar graphs of representative genes (Figures 5J and 5M) (mean ± SEM expression across all cells in that cluster, with each bar graph scaled to the maximum mean expression across all clusters), and in-situ spatial distribution of excitatory (Figures 5I-5K) and inhibitory (Figures 5L-5N) neurons (Figures 5K and 5N). The number of cells in each cluster was as follows: L2 / 3: 589; L4: 649; L5: 393; L6: 368; PV neurons: 111; VIP neurons: 46; Sst neurons: 46; Npy neurons: 56. Cell inclusion within clusters was completely induced by the amplicon expression within each cell without using spatial information. Then, excitatory cell clusters were named according to the spatial layer observed for that cluster, while inhibitory cell clusters were named according to the dominant cell type amplicon based on the strong separation of amplicon markers. 【0168】 Figure 6C illustrates a method for clustering and subclustering expression data per cell, where (1) the data is represented in a cell × gene matrix as z-scored log-transformed counts, (2) principal component analysis (PCA) is applied to the matrix to reduce it to a cell × coefficient matrix, (3) cell locations are plotted using Uniform Manifold Approximation (UMAP) (a nonlinear dimensionality reduction technique for 2D visualization of high-dimensional data) for visualization, (4) cells are clustered by PCA values ​​using shared nearest neighbor-based graph clustering, and (5) expression values ​​for cells corresponding to individual clusters are then obtained and used again for subclustering. 【0169】 Richly defined excitatory neurons were separated into four major types (eL2 / 3, eL4, eL5, and eL6; Figures 5I-5K and 7A-7B) indicated by spatial correspondence with anatomical cortical layers and expression profiles of known layer-specific gene markers. The Z-scored expression matrix of excitatory cell types in Figure 7A showed clustering of numerous differentially expressed genes per cell type. The genes shown were analyzed using likelihood ratio tests for the expression of the gene in any other cell within each cluster. -10 Selection was based on a P-value threshold adjusted for the false detection rate (FDR) and a minimum log10 multiplier change of 0.1. Figure 7B depicts a UMAP visualization of the relative expression (normalized to minimum and maximum across all excitatory cells) of numerous known layer-specific genes enriched in each cluster across cells, showing that most are enriched in specific excitatory subtypes. Figure 7C depicts the expression matrix of inhibitory cell types selected as in Figure 7A. Figure 7D depicts a UMAP visualization showing the relative expression of known interneuronal marker genes, showing that each is specifically enriched in inhibitory neuronal subtypes. 【0170】 The spatial configuration of the four excitatory types exhibited a layered pattern, but there was extensive mixing among the different cell types within each layer. Inhibitory neurons were also clustered into four major types (Vip, Sst, Npy, and Pvalb, Figures 5L–5N and 7C–7D) indicated by dominant interneuronal markers for each subtype. Vip and Npy types were observed to be distributed more in the upper layers (L1–3), while Sst and Pvalb types were more commonly found in the lower layers (L4–6). 【0171】 Non-neuronal cell types, including astrocytes, oligodendrocytes, endothelial cells, and smooth muscle cells, were also detected (Figures 8A-8C). The number of major cell types exemplified here (12 in total) can be further broken down (single-cell RNA sequencing can result in more than 40 subtypings, consistent with the readily apparent heterogeneity of gene expression within each type; Figures 7A-7D; Figures 8A-8C). UMAP visualizations of four non-neuronal cell types are depicted in Figure 8A. The Z-scored expression matrices of the non-neuronal cell types are depicted in Figure 8B. The genes shown were expressed using likelihood ratio tests for the genes expressed in cells within each cluster versus cells in any other cluster. -10 Selection was based on a P-value threshold adjusted for the false detection rate (FDR) and a minimum log10-fold change of 0.1. Figure 8C depicts the UMAP visualization of per-cell expression of marker genes (top differentially expressed genes per cluster) for non-neuronal cell types, showing specificity for that cluster. The colors indicate the relative expression (normalized to minimum and maximum) of each gene across all non-neuronal cells. 【0172】 Using a set of 112 targeted genes, all 12 major cell types were reliably detected in four biological replications with very similar spatial patterns and without batch effects, at cell sizes of 600–800 cells per sample (Figures 9A–9G). Figures 9A–9C depict UMAP visualizations of cells encoded by sample replication, then grouped by major clusters (Figure 9A), excitatory subclusters (Figure 9B), and repressive subclusters (Figure 9C). Spatial maps of pairs of light-replication and dark-replication (Figures 9D–9G, top and bottom) for all cell types (Figure 9D), excitatory cell types (Figure 9E), repressive cell types (Figure 9F), and non-neuronal cell types (Figure 9G). 【0173】 Example 6: Single-cell RNA sequencing The quantitative capabilities of STARmap at the single-cell level were evaluated to test differential gene expression analysis across experimental conditions in molecularly defined cell types. Visual stimulus-dependent gene expression patterns (via 48 defined ARGs with single-cell resolution in situ) were assessed. Further development of the single-cell RNA sequencing procedure, mouse brains were flash-frozen with minimal post-sacrifice handling time (less than 5 minutes) and without drug treatment for maximum preservation of intrinsic transcriptional signatures. Global induction of known early genes (Fos, Egr1, and Egr2, Figures 10A–10D) was observed in the primary visual cortex after 1 hour of light exposure. At single-cell resolution, the quantitative range of ARG changes (multiplicative changes in expression) showed remarkable diversity across neuronal cell types (Figures 10B–10C and 11A–11C). Figure 10A examines the spatial expression patterns in the visual cortex of prototypical ARGs known as early genes (IEGs). Sacrifice was performed in darkness or after 1 hour of light exposure. Figures 10B and 10C depict volcanic plots of logarithmic changes in gene expression between light and dark conditions in repressive and excitatory cell types. Genes with significantly increased or decreased expression (P-values ​​< 0.05, adjusted for false-find rate in Wilcoxon rank-sum test) are labeled green, and the most significantly changed genes (P-values ​​< 0.05 and magnification change > 2) are labeled red. Many ARGs showed cell type specificity, indicating the discovery of an unexpected cell type-specific logic in excitation-transcription coupling. Figure 10D depicts violin plots of Egr2 expression by cell type. **** indicates P < 0.0001, ns indicates not significant in Wilcoxon rank-sum test, and the magnification change for red-labeled cell types was greater than 2. In general, ARG expression programs in excitatory neurons across different layers were very similar, but ARG expression programs in inhibitory cells exhibited much different cell type-specific characteristics (Figure 11B). For example, Egr2 showed photoinduction across excitatory neurons but not in inhibitory neurons (Figure 10D), while in contrast, Prok2 was upregulated in Vip inhibitory neurons (Figure 10C). 【0174】 Finally, since neuronal activity can induce co-transcription of non-coding RNAs from within ARG enhancers, examples of these enhancer RNAs were studied (eRNA1-5 of the Fos gene), and these unpolyadenylated transcripts were extremely difficult to measure with current single-cell RNA sequencing. However, eRNA3 was identified as the most significant and consistent ARG marker (Figure 11B). Figure 11A depicts the correlation of 160 genes for dark / photobiological replication, showing that samples under the same conditions correlated more highly with scale and Spearman R values ​​than samples under different conditions. Figure 11B depicts logarithmic scale expression data (number per cell) of ARGs in inhibitory and excitatory neuron subtypes. Genes with significantly increased expression in any cell type are highlighted in red. Figure 11C depicts a heatmap of neural subtype correlations based on the correlation of mean expression of all ARGs in that cluster in response to light, showing that cells from inhibitory cell types correlated more with other inhibitory cell types than with excitatory cell types, and vice versa. Note that the scale ranged from R=0.8 to R=1. 【0175】 Example 7: STARmap method applied to large tissue volumes Since 160 gene experiments were performed on brain sections less than the thickness of a single cell body, the ability of the STARmap method to capture the 3D configuration of cells in tissue volume was not yet tested. STARmap has been further developed to overcome limitations in diffusion access to intact tissue volume and imaging throughput using strategies to enable high-throughput molecular analysis in tissue volume by linearly reading gene expression at cell resolution (Figures 12 and 13). Figure 12 depicts the method disclosed herein, including the steps of sample sizing, library preparation, imaging and sequencing, and data output. For thin tissues (z<16 μm, cell monolayer), libraries were prepared using fresh-frozen mouse brain, followed by cryostat slicing, PFA fixation, permeabilization, hybridization, ligation and amplification, as well as hydrogel embedding and tissue clarification. Imaging and sequencing included single amplicon resolution, high NA oil immersion objective lenses, imaging of 200 cells per hour, SEDAL reaction with degenerate probes, and exponential readout from cycles to genes. Data output included 3D amplicon and 2D cell typing (Figures 1A–1E, 5A–5N, and 10A–10D). For thicker tissues (z > 100 μm, numerous cell layers), libraries were prepared using PFA-fixed mouse brain, followed by vibratome slicing, clearing and hybridization, hydrogel embedding and tissue clarification, as well as ligation and amplification. Imaging and sequencing included single-cell resolution, low NA water immersion objectives, imaging of 10,000 cells per hour, SEDAL reactions with orthogonal probes, and linear readout from cycles to genes. Data output included 3D cell typing (Figures 13A–13I). 【0176】 Figure 13A depicts volumetric STAR mapping via sequential SEDAL gene readout. A modified STARmap procedure (Figure 12, right) and circular gene readout (four genes in each cycle) were used to rapidly map large tissue volumes at single-cell resolution without oversampling each amplicon. Using Thy1::YFP mouse brain, the specificity and penetration depth of large-volume STARmap were initially tested. STARmap successfully detected YFP mRNA across a tissue thickness of 150 μm, and notably co-localized YFP protein and mRNA at single-cell resolution without labeling tens of thousands of scattered adjacent cells (Figure 13B). Validation showed specific STARMAP labeling of YFP-expressing neurons (from the transgenic Thy1::YFP mouse strain) in 3D cortical volume. Scale bar indicates 0.5 mm. 【0177】 Spatial cell typing of the mouse primary visual cortex was extended to over 30,000 cells across the volume spanning all six layers and the corpus callosum. Using 23 cell type markers and 28 genes, including 5 ARGs read over 7 cycles of linear SEDAL sequencing (Figures 13C–13D and 14A–14B), K-mean clustering of marker genes was applied to each cell type (11 cell types were recovered corresponding to the majority of those extracted by 160 gene experiments, but only 28 genes are shown here). Figure 13C depicts representative markers of major cell types (Figure 13C, left), layer-specific markers (Figure 13C, left center), inhibitory markers (Figure 13C, right center), and activity-regulated genes (Figure 13C, right) obtained over numerous rounds in the visual cortex STARmap volume. 【0178】 3D patterning of 11 cell types (Figures 13E–13F) was consistent with the histological findings of 160 gene sections, but provided accurate and quantitative profiling of the spatial cell distribution with a much larger number of cells. Spatial histograms of excitatory, inhibitory, and non-neuronal cell types were created using the same labeling as in Figure 13D (Figure 13E). Cells were counted in 5 μm bins at 2D maximum projection and plotted in units of cell count / μm as a function of distance from the corpus callosum (cc) to the pia mater, and averaged across bins perpendicular to the cortical layers. Plots of maximum projected cell locations were coded by clusters as in Figure 13D (Figure 13E, bottom). As reflected by both the spatial histograms (Figure 13E) and correlation analysis (Figure 14B), the excitatory subtypes exhibited a layered distribution, with the spatial density of each subtype attenuating spatially to adjacent layers. In contrast, the repressive subtypes were more dispersed, despite the tier preference tendencies indicated by the Vip subtype (primarily located in tiers 2 / 3), as well as the Sst and Pvalb subtypes (located in tiers 4 and 5). Figure 14A depicts the expression of each gene in 3D maxima projected onto the XY plane and shows the spatial distribution of gene expression values ​​extracted per cell, which will be used for subsequent clustering. Each gene was z-scored cell-wise across all genes. Figure 14B depicts the voxel-level correlation coefficients between the distributions for each cell type, binned on a 25 μm grid. 【0179】 Non-neuronal cells were primarily observed in layer 1 and the white substance. Figure 13D depicts the per-cell expression matrix of 28 genes from 32,845 single cells clustered into numerous excitatory, inhibitory, and non-neuronal cell types, z-scored across genes for each cell to normalize with respect to the mean difference in overall signal between cells. The columns were sorted in order of sequencing rounds performed, in four groups. Figure 13F depicts the spatial distribution of each cell type (excitatory, inhibitory, and non-neuronal) and subtype in three dimensions. Each dot represents a single cell, and the spatial dimension was μm. 【0180】 To discover finer volume patterns, we analyzed the distribution of distances from individual cells of each defined subtype to their nearest neighbors and unexpectedly found that the nearest neighbors of any inhibitory neuron tended to be of its own subtype, rather than excitatory neurons or other inhibitory subtypes (Figure 13G). Figure 13G depicts the nearest neighbor distances calculated in 3D between all excitatory cells (excitatory) and each inhibitory cell type. For self-comparison, nearest neighbor was defined as the closest non-identical cell, and persistent autocorrelation revealed self-clustering of inhibitory subtypes. If inhibitory neurons were randomly dispersed among more abundant excitatory neurons in a pure salt-and-pepper distribution, the distances between inhibitory neurons would be greater than the distances from inhibitory neurons to excitatory neurons (Figure 13H). As shown in Figure 13H, we analyzed the same distances but used shuffled (randomized) cell type labeling. The actual intra-subtype distances of inhibitory neurons were much shorter (approximately 15 μm, equal to the size of a single neuron, indicating direct somatic cell juxtaposition; Figure 13I). Figure 13I depicts the 3D nearest neighbor distance calculated between each inhibitory cell of a particular type and any member of the same type (inhibitory-inhibitory, e.g., VIP-VIP) or any excitatory neuron (inhibitory-excitatory), where **** indicates P < 0.0001 by Wilcoxon rank-sum test. Figure 13I reveals the self-clustering configuration of inhibitory subtypes across volume, which could only be accurately measured in 3D and not 2D (Figures 15A-15C). The mean nearest neighbor distance between excitatory cell types and different inhibitory cell types, calculated in 2D projections of 8 μm (thinner than a single cell) sections along the Z direction, was obtained within the same 3D volume shown in Figures 13A-13I (Figure 15A). The 2D nearest neighbor distance could not accurately estimate the 3D distance for the same cell types shown in Figure 13D (overestimation). Such patterns may be functionally related; for example, in vivo imaging suggests that inhibitory neuron clusters in the visual cortex can sharpen visual responses. Figure 15B depicts an example of a 3D short-range cluster of inhibitory neurons, a zoomed-in view from Figure 13C.Figure 15C depicts a short-range inhibitory neuron cluster (generated by crossing Parv-IRES-Cre with Ai14) observed in the primary visual cortex of transgenic mice. Pvalb cells were labeled with tdtomato, and all neuronal nuclei were immunostained with Alexa647-conjugated anti-NeuN. 【0181】 Example 8: STARmap Scalability STARmap adapted to longer sequencing lengths or higher gene counts, and there were no inherent limitations on the number of genes or RNA species that could be quantitatively accessed simultaneously by STARmap (Figures 16A–16E). Figure 16A depicts the detection of Actb mRNA when co-detected with increasing amounts of other RNAs to test the potential dilution effect of probe mixtures, along with the physical capacity of cells for SNAIL DNA amplicons. SNAIL probes for Actb were designed with orthogonal DNA sequences for detection and spiked into mixtures with probes for 0 (Figure 16A), 100 (Figure 16B), or 1,000 (Figure 16C) of other genes. When the SNAIL probe acted in a mixture rather than as a single probe, if the efficiency was lower or there was not enough space for rolling circle amplification, Actb spike-in resulted in fewer amplicons and / or a reduced intensity for each amplicon. Fluorescence images were acquired in the mouse visual cortex, where green represents the Alexa546 channel of the Actb amplicon, red represents the Alexa647 channel of all other genes, and blue represents DAPI staining of the cell nucleus. Quantifications from Figures 16A–16C are depicted in Figure 16D. The box plots show that neither dilution nor cell space limitation has a significant effect up to a scale of at least 1,000 genes. The boxes represent the first and third quartiles, the median line represents the median, the whiskers represent the 5% and 95% data points, n represents the number of Actb amplicons across a 228 × 228 × 2 μm imaging volume, and the y-axis represents absolute fluorescence intensity. Figure 16E depicts experimental and theoretical estimates of STARmap's scalability. The coding shows that 5nt of coding can encode 1,024 genes, and the SNAIL probe has 35nt of coding space in addition to the RNA complementary sequence, while SEDAL requires 17nt as a sequencing unit (11nt docking region for the reading probe + 5nt coding and 1nt flanking base), so the SNAIL probe holds two such units, and 4 10 (10 6) allows for more codes, and other sequencing methods for longer reads (e.g., SOLiD, 18nt for primer binding and 17nt for coding) have an upper limit of 10 11 It approached the number of individuals. The physical capacity was examined for up to 1,000 genes in mammalian neurons, and since the physical size of the DNA amplicons was about 100-200 nm as determined by AFM and TEM, and considering that the cell diameter was about 15 μm and a closed model (space efficiency 74%) was used, the estimated maximum capacity was 10 per cell. 6 There were 2 × 10¹ amplicons. Optical volume was verified in 1,020 gene experiments in mouse hippocampal cell cultures and visual cortex experiments, with amplicon / cell referring to those successfully aligned throughout all 6 sequencing rounds. As imaged by confocal microscopy, the average diameter of DNA amplicons was 400–600 nm, and applying the same model used for physical limits, the maximum volume was 2 × 10¹ cells. 4 There were amplicons. Experimental data for 1,020 genes approached this limit. While not bound by any scientific theory, the numerical differences between cell cultures and tissue sections may be due to the following considerations: (1) whole cells were imaged in the cell culture while cell fractions were imaged in the tissue section (8 μm, < thickness of one cell); (2) hippocampal cell cultures are less differentiated compared to adult mouse brains and therefore show more RNA diversity per cell (more genes); (3) in vivo cultured cells were considerably more spread out in the xy plane and thinner in the z direction, in contrast to the high-density 3D packing of cells in brain tissue, but the images in the xy plane showed higher optical resolution compared to the z direction (voxel size 78 × 78 × 250 μm). 【0182】 Example 9: Correlation between neuron types identified in 160 gene experiments using STARmap and publicly available single-cell RNA sequencing results. Figure 17 depicts the Pearson correlation between average gene expression across all genes in the identified excitatory and repressive clusters of the STARmap and the corresponding clusters identified by single-cell RNA-seq from the Allen Brain Institute. See, for example, Lein et al. (2007). For single-cell RNA-seq data, expression was averaged across all subtypes within the major type (e.g., L2 / 3), and correlations were calculated using only genes common between single-cell RNA-seq and the 160-gene V1 experiment, with a scale of 0–0.6 on the Pearson correlation coefficient. 【0183】 Example 10: Gene expression analysis of cell type subclusters in the medial prefrontal cortex (mPFC) using STARmap Figure 18A depicts the UMAP visualization of excitatory subclusters. Figure 18B depicts the differentially expressed genes per excitatory subcluster. Figure 18C depicts the UMAP visualization of repressive subclusters. Figure 18D depicts the differentially expressed genes per repressive subcluster. Figure 18E depicts the UMAP visualization of non-neuronal subclusters. Figure 18F depicts the differentially expressed genes per non-neuronal subcluster. 【0184】 Figures 19A–19C depict spatial maps across four biological replications: excitatory subclusters (Figure 19A), inhibitory subclusters (Figure 19B), and non-neuronal subclusters (Figure 19C). 【0185】 Example 11: Gene expression analysis of 1020 genes in cell type subclusters in mouse hippocampal cell cultures Figures 20A–20C depict the analysis of 1020 genes in mouse hippocampal cell cultures in six rounds of sequencing using the methods described herein. Figure 20A depicts the fluorescence image of faw integrating four fluorescence channels in the first round. Figure 20B depicts an example of cell type markers. Neuronal gene markers (Scna) are well separated from non-neuronal gene markers (Mt1), and the distribution of neuronal subtype markers (Reln, Sst) is different. Figure 20C depicts the statistical analysis of amplicons and genes per cell in an imaging area of ​​270 × 270 μm. 【0186】 Figures 21A–21C depict additional gene expression information for 1020 genes in the mouse primary visual cortex using the methods described herein. Figure 21A depicts UMAP visualizations of excitatory subclusters (Figure 21A, left) and differentially expressed genes per excitatory subcluster (Figure 21A, right). Figure 21B depicts UMAP visualizations of repressive subclusters (Figure 21B, left) and differentially expressed genes per repressive subcluster (Figure 21B, right). Figure 21C depicts UMAP visualizations of non-neuronal subclusters (Figure 21C, left) and differentially expressed genes per non-neuronal subcluster (Figure 21C, right). 【0187】 Figures 22A–22D depict the reproducibility of 1020 genes in the mouse primary visual cortex using STARmap, and cross-method comparisons of the measurements. Figure 22A depicts the correlation of per-gene loadings between two 1020-gene replications in the visual cortex. Figure 22B depicts histograms of detected loadings per cell (Figure 22B, left) and detected genes per cell (Figure 22B, right). Figure 22C depicts a spatial map of cell types in other replications of the 1020 genes in the visual cortex experiment. Figure 22D depicts the Pearson correlation between mean gene expression across all genes in the identified 1020-gene clusters of STARmap and the corresponding clusters identified by single-cell RNA-seq from the Allen Brain Institute. 【0188】 Example 12: STARmap for thin and thick tissue sections Figure 23A illustrates experimental flowcharts for STARmap for thin and thick tissues. Figure 23B shows the preparation of modified primer probes for large-scale experiments. DNA probes were ordered with 5' amine modifications, pooled, and converted to polymerizable moieties by AA-NHS. Figure 23C shows the experimental duration for different experimental designs using varying numbers of genes. Figure 23D shows a comparison of STARmap's RNA species, spatial resolution, and throughput with other single-cell approaches. Single-cell RNA sequencing, when combined with recently developed spatial transcriptome methods, can achieve regional spatial resolution (100 μm). 【0189】 While the present invention is described with reference to its specific embodiments, it should be understood by those skilled in the art that various modifications may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt specific circumstances, materials, composition of substances, processes, process steps, or steps to the object, spirit, and scope of the invention. All such modifications are intended to be within the scope of the claims appended herein. References 1.N.Crosetto,M.Bienko,A.van Oudenaarden,Spatially resolved transcriptomics and beyond.Nat.Rev.Genet.16,57-66. 2.E.Lein,LEBorm,S.Linnarsson,The promise of spatial transcriptomics for neuroscience in the era of molecular cell typing.Science 358,64-69(2017). 3. E. 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Claims

[Claim 1] A method for identifying target nucleic acids in cells within fixed and permeabilized intact tissue, wherein the method is: (a) Contacting the immobilized, permeabilized intact tissue with the pair of oligonucleotides under conditions that enable specific hybridization of the pair of oligonucleotides to the target nucleic acid, wherein the pair of oligonucleotides are (i) A first oligonucleotide comprising a first complementary region, a second complementary region, and a third complementary region, (ii) A second oligonucleotide comprising a fourth complementary region, a fifth complementary region, and a sixth complementary region, and Including; The first complementary region is complementary to the first portion of the target 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 the second portion of the target nucleic acid, and the first complementary region is adjacent to and in contact with the fifth complementary region. (b) Performing an amplification reaction on the target nucleic acid having the pair of oligonucleotides hybridized thereto to produce one or more amplicons, (c) embedding one or more amplicons in a hydrogel, (d) Image one or more amplicons to identify the target nucleic acid, Methods that include... [Claim 2] The method according to claim 1, wherein (c) comprises clarifying the fixed and permeabilized intact tissue among a plurality of cellular components. [Claim 3] The method according to claim 2, wherein the cellular component comprises lipids, proteins, or a combination thereof. [Claim 4] The method according to claim 1, wherein the pair of oligonucleotides are denatured by heating before (a). [Claim 5] The method according to claim 1, wherein the cells are located within a group of cells. [Claim 6] The method according to claim 5, wherein the population of cells includes a plurality of cell types. [Claim 7] The method according to claim 1, wherein the fixed and permeabilized intact tissue is fixed and permeabilized with a single solution. [Claim 8] The method according to claim 1, wherein the target nucleic acid is ribonucleic acid (RNA). [Claim 9] The method according to claim 1, wherein the target nucleic acid is messenger RNA (mRNA). [Claim 10] The method according to claim 1, wherein the target nucleic acid is deoxyribonucleic acid (DNA). [Claim 11] The method according to claim 1, wherein the second oligonucleotide is provided as a closed nucleic acid ring. [Claim 12] (i) The second oligonucleotide comprises a padlock probe, (ii) The first complementary region of the first oligonucleotide has a length of 19 to 25 nucleotides, (iii) The second complementary region of the first oligonucleotide has a length of six nucleotides, (iv) The third complementary region of the first oligonucleotide has a length of six nucleotides, (v) The fourth complementary region of the second oligonucleotide has a length of six nucleotides, (vi) The fifth complementary region of the second oligonucleotide has a length of 19 to 25 nucleotides. (vii) The sixth complementary region of the second oligonucleotide has a length of six nucleotides, (viiii) The fourth complementary region of the second oligonucleotide includes the 5' end of the second oligonucleotide. (ix) The sixth complementary region of the second oligonucleotide includes the 3' end of the second oligonucleotide. (x) The fourth complementary region of the second oligonucleotide is adjacent to the sixth complementary region of the second oligonucleotide, or (xi) The barcode sequence of the second oligonucleotide provides barcode information for identifying the target nucleic acid. The method according to claim 1. [Claim 13] The method according to claim 1, wherein (d) image the one or more amplicons using a microscopy selected from the group consisting of confocal microscopy, wide-field fluorescence microscopy, two-photon microscopy, bright-field microscopy, intact tissue expansion microscopy, super-resolution microscopy, and light-sheet microscopy. [Claim 14] The method according to claim 1, wherein the fixed and permeable intact tissue has a thickness of 5 to 20 micrometers (μm). [Claim 15] The method according to claim 1, wherein the fixed and permeable intact tissue has a thickness of 50 to 200 micrometers (μm). [Claim 16] A method for screening candidate drugs and determining whether the candidate drugs regulate the gene expression of the target nucleic acid in cells within intact tissue, the method comprising performing the method according to claim 1 to determine the gene sequencing of the target nucleic acid in the cells within the intact tissue, The detection of the level of gene expression of the target nucleic acid, wherein the change in the level of expression of the target nucleic acid in the presence of the at least one candidate drug compared to the level of expression of the target nucleic acid in the absence of the at least one candidate drug indicates that the at least one candidate drug modulates the gene expression of the nucleic acid in the cells within the intact tissue. Methods that include... [Claim 17] The method according to claim 1, further comprising adding a ligase before (b) to ligate the second oligonucleotide and generate a closed nucleic acid ring. [Claim 18] The method according to claim 17, wherein the ligase is a DNA ligase. [Claim 19] The method according to claim 1, wherein the amplification reaction comprises rolling circle amplification, wherein the second oligonucleotide is a template and the first oligonucleotide is a primer for a polymerase to form the one or more amplicons. [Claim 20] The method according to claim 1, wherein (c) comprises copolymerizing one or more amplicons with acrylamide.

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