Signal amplification methods and compositions for molecular target detection by iterative probe deposition

Abstract

The present disclosure provides methods for scalable amplification of an analyte signal in a sample to achieve spatial localization of the analyte in a biological sample. The present disclosure sets forth the methods, as well as the use of these methods and solutions to other issues in the related art.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 524,408, filed June 30, 2023. The entire contents of the above-cited application are hereby incorporated herein by reference.

[0003] Statement regarding federally funded research or development

[0004] The U.S. government has certain rights in this disclosure, pursuant to grant NS131408 from the National Institutes of Health. Technical Field

[0005] This disclosure provides methods, compositions, and kits for scalably amplifying analyte signals in samples to spatially locate the analyte in biological samples. The invention can be implemented for multiplex analysis of one or more types of analytes in biological or clinical samples, such as, but not limited to, RNA, DNA, proteins, small molecule inhibitors, sugars, lipids, organelles, and synthetic barcodes. Background Technology

[0006] Spatial analysis of cellular components, including DNA regions, proteins, and mRNA, plays a crucial role in understanding cellular function and the molecular mechanisms underlying health and disease. Microscopy, capable of resolving these targets within single cells, provides key insights into the abundance and localization of these components.

[0007] Spatial transcriptomics has become a powerful tool for understanding the spatial organization of gene expression in tissues and cells, providing valuable insights into cellular heterogeneity and tissue function (1). A major limitation of imaging-based spatial transcriptomics methods is the low signal-to-noise ratio, especially in thick tissue samples or non-ideal tissue samples such as FFPE or human tissue samples. Typically, weak signals can be circumvented by using a greater number of hybridization probes per gene. However, this approach is ineffective when targeting short RNA species.

[0008] Several methods have been developed to address these challenges, such as ClampFISH (2, 3), RollFISH (4), STARmap (5, 6), HybISS (reference 7), FISSEQ (8), hybridization chain reaction (9, 10), and branched DNA (11, 12). However, these methods typically exhibit several drawbacks, such as poor probe diffusion, nonspecific signals, low detection efficiency, inability to scale to near-transcriptome levels, or the need for extensive screening of amplification probes. Most of these methods require enzymes, which can be costly and require special storage conditions; they rely on highly purified and bifunctional probes, which can also be costly and time-consuming to generate; they depend on unstable reagents, which may limit reproducibility and scalability; or they require pre-programmed probes to be assembled at defined amplification folds, which does not provide modularity for the overall approach. Summary of the Invention

[0009] This disclosure provides a method for scalably amplifying analyte signals in a sample to spatially locate the analyte in a biological sample. This disclosure describes the method, as well as other solutions to problems in the related art using these methods.

[0010] In some embodiments, a method is provided for scalably amplifying an analyte signal in a sample to spatially localize the analyte in a biological sample, comprising: contacting one or more target analytes in the sample with a plurality of primary probes, wherein each of the plurality of primary probes interacts with at least one target analyte. In some embodiments, the method comprises: contacting each of the plurality of primary probes with one or more amplification probes, each primary probe interacting with at least one analyte. In some embodiments, the method comprises: optionally, crosslinking one or more amplification probes to a cellular component. In some embodiments, the method comprises: optionally, separating each of the one or more amplification probes from its primary probe or from another amplification probe. In some embodiments, the method comprises: optionally, repeating any of the foregoing embodiments individually or in combination. In some embodiments, the method comprises: detecting one or more target analytes.

[0011] In some embodiments, the method includes contacting each of a plurality of amplification probes with one or more readout probes. In some embodiments, the method includes imaging a sample to detect the interaction between the readout probes and their target analytes.

[0012] In some implementations, the method includes imaging the sample after contacting the amplification probe with one or more readout probes.

[0013] In some embodiments, the method further includes amplifying the amplification probe by contacting the amplification probe from a previous contact step with a plurality of new amplification probes.

[0014] In some embodiments, the method further includes: repeating the steps of contacting the amplification probe with one or more readout probes and the imaging step, each time using a new plurality of readout probes, such that the target analyte is described by a barcode and can be distinguished from another target analyte in the sample by the difference in its barcode. In some embodiments, the method includes: optionally, separating the amplification probe from the primary probe or another amplification probe after imaging the sample. In some embodiments, the method includes: optionally, separating the readout probe from the amplification probe.

[0015] In some embodiments, a method is provided for scalably amplifying an analyte signal in a sample to spatially localize the analyte in a biological sample, comprising: contacting one or more analytes with primary probes of a plurality of primary probes, wherein each of the plurality of primary probes interacts with at least one target analyte. In some embodiments, the method includes: contacting each primary probe interacting with at least one analyte with one or more amplification probes. In some embodiments, the method includes: crosslinking the amplification probes to a cellular target. In some embodiments, the method includes: separating the amplification probes from the primary probes. In some embodiments, the method includes: optionally, repeating any of the foregoing steps. In some embodiments, the method includes: contacting each amplification probe with one or more readout probes. In some embodiments, the method includes: imaging the sample after contacting the amplification probes with one or more readout probes to detect the interaction between the readout probes and their target analytes.

[0016] The methods detailed in this disclosure provide substantially more scalable, cost-effective, and modular solutions. We provide a method in which the amplification fold can be modularly adjusted in situ, utilizing easily functionalizable and moderately pure oligonucleotides, and providing a non-enzymatic solution for in situ amplification. Attached Figure Description

[0017] Figure 1 An exemplary design for a linear amplification process is described. Figure 1 Figure A illustrates an exemplary implementation of the binding of a primary probe to a target molecule. Figure 1 Figure B illustrates an exemplary implementation where the amplification probe is combined with a unique region of the primary probe. Figure 1 Figure C illustrates an exemplary implementation where the amplification probe binds to the sample via a reactive group. Figure 1 Figure D illustrates an exemplary implementation of the binding of subsequent amplification probes with primary probes. Figure 1E illustrates an exemplary implementation of multiple rounds of amplification probe binding to a primary probe, binding to a sample, and dissociating from the primary probe to allow the next amplification probe to bind to the same primary probe site. Figure 1 Figure F illustrates an exemplary implementation of a readout probe combined with an amplification probe bound to a sample to detect target molecules.

[0018] Figure 2 An exemplary design for a process that does not bind the probe to the sample is described. Figure 2 Figure A illustrates an exemplary implementation of the binding of a primary probe to a target molecule. Figure 2 Figure B illustrates an exemplary implementation of the binding of the amplification probe to the primary probe binding region. Figure 2 Figure C illustrates an exemplary implementation where the amplification probe does not bind to the sample. Figure 2 Figure D illustrates an exemplary implementation where the readout probe does not bind to the sample, as shown by the dashed line.

[0019] Figure 3 An exemplary design for a segmented linear amplification process is described. Figure 3 Figure A illustrates an exemplary implementation of multiple amplification probes binding to a sample. Figure 3 Figure B illustrates an exemplary implementation where a secondary amplification probe contacts a primary probe bound to a sample. Figure 3 Figure C illustrates an exemplary implementation of binding a secondary amplification probe to a sample. Figure 3 Figure D illustrates an exemplary implementation of adding more secondary amplification probes during subsequent rounds of secondary amplification probe addition, sample binding, and dissociation from the initial amplification probe. Figure 3 E illustrates an exemplary implementation of adding more secondary amplification probes during a subsequent round of secondary amplification probe addition, sample binding, and dissociation from the initial amplification probe. Figure 3 Figure F illustrates an exemplary implementation in which each secondary amplification probe can be bound to a labeled readout probe, which can be used to detect molecular targets in a sample.

[0020] Figure 4 Exemplary designs for linear and exponential amplification processes are depicted. Figure 4 Figure A illustrates an exemplary implementation of multiple amplification probes binding to a sample. Figure 4 Figure B illustrates an exemplary implementation where a secondary amplification probe contacts a primary probe bound to a sample. In this example, each amplification probe used in an exponential amplification round has two binding sites for the next round of amplification probes. Figure 4 Figure C illustrates an exemplary implementation of binding an exponential amplification probe to a sample. In this example, the amplification probe has two binding sites for the next round of amplification probes. Figure 4Figure D illustrates an exemplary implementation of binding exponential amplification probes to a sample, wherein two amplification probes bind to each amplification probe bound in the previous round. In this example, an amplification probe applied in the second round of exponential amplification can bind to an amplification probe applied in the first round, which in turn can bind to an amplification probe applied in the second round, and so on.

[0021] Figure 5 An exemplary design is described that follows an exponential amplification process based on the amplification factor according to the Pell sequence. Figure 5 Figure A illustrates exemplary implementations of primary probe binding to target molecules and amplification probe binding to primary probes. Figure 5 Figure B illustrates an exemplary implementation in which two secondary amplification probes bind at feature A- to a cross-linked and cleaved first amplification probe at feature A+. Figure 5 Figure C illustrates an exemplary implementation of the next round, in which the same amplification probe as in the initial round is applied to the sample. Figure 5 Figure D illustrates an exemplary implementation of another round of amplification using a secondary amplification probe, such as... Figure 5 B describes binding to the A+ site. Figure 5 Figure E illustrates an exemplary implementation of the next round of amplification, in which the first amplification probe is added to the sample again. Combined with similar... Figure 5 As stated in C.

[0022] Figure 6 An exemplary design for an exponential amplification process without cutting or displacement is described. Figure 6 Figure A illustrates an exemplary implementation of the binding of a primary probe to a target molecule and the binding of an amplification probe to a primary probe. The amplification probe components labeled in this example are: an R- binding site for the primary probe, two A+ binding sites for subsequent amplification probe binding, and a connectable feature that can bind, for example, to the sample. Figure 6 Figure B illustrates an exemplary implementation where two subsequent amplification probes bind to the amplification probe bound to the primary probe in the previous figure. In this figure, the ligation-capable feature of the amplification probe is stably bound to the sample. The amplification probe components marked in this example are: a binding site A- for the previous amplification probe binding site A+, two R+ binding sites for subsequent amplification probe binding, and the ligation-capable feature. Figure 6 Figure C illustrates an exemplary implementation where two subsequent amplification probes bind to an amplification probe from the previous round. The amplification probes in this example have... Figure 6 The same components are used in round 1 as shown in Figure A. Additional exponential amplification rounds exceeding the three rounds shown in this exemplary embodiment increase the number of amplified probes binding near the primary probe binding site.

[0023] Figure 7An exemplary design of an amplification probe that can be bound to a sample is described. Figure 7 Figure A illustrates an exemplary implementation of a primary probe that binds to an amplification probe. Figure 7 Figure B illustrates something similar to Figure 7 Example A illustrates an exemplary implementation of the amplification probe, but it shows two binding sites A+ instead of one binding site for designs involving, for example, stronger linear or nonlinear amplification schemes. The cleavage or displacement features included in this example figure may be dedicated portions of the probe, or may partially or completely overlap with the A+ example binding site of the adjacent R-primary probe binding site for various purposes, including displacement, cleavage, and / or amplification and readout of the probe binding site.

[0024] Figure 8 An example design of a scheme using photocrosslinking and displacement of the probe from the primary probe is depicted. Figure 8 Figure A illustrates exemplary implementations of primary probe binding to target molecules and amplification probe binding to primary probes. Figure 8 Figure B illustrates an exemplary implementation showing a more detailed view of the binding of the amplification probe to the primary probe at the R3+ / R3- site. The crossed circles in this example indicate that the amplification probe binds to the sample via a connectable feature. Figure 8 Figure C illustrates an exemplary implementation of the binding of a displacement probe and an amplification probe. In this example, binding is aided by the stalk feature T-+ on the amplification probe, which makes the binding of the displacement probe more favorable than the binding of R3- to R3+ on the primary probe. Figure 8 Figure D illustrates an exemplary implementation where the replacement probe remains attached to the amplification probe after excess replacement probe is washed away. The replacement probe remains on the amplification probe to prevent the amplification probe from rebinding to the primary probe. Figure 8 E illustrates an exemplary implementation of a subsequent amplification cycle, in which another amplification probe binds to the R3+ feature of the primary probe and binds to the sample via a connectable feature (circle). The number of amplification cycles used for linear signal amplification is shown above the example image.

[0025] Figure 9 An example design of a scheme using photocrosslinking and displacement of the probe from the primary probe is described, wherein the permutation portion binds to a second readout site on the amplification probe. Figure 9 Figure A illustrates exemplary implementations of primary probe binding to target molecules and amplification probe binding to primary probes. Figure 9 Figure B illustrates an exemplary embodiment of the amplification probe binding to the primary probe at the R3+ / R3- site in a more detailed view. In this exemplary embodiment, the amplification probe has two A3+ amplification probe binding sites instead of a dedicated stalk sequence. The crossed circles in this example indicate that the amplification probe binds to the sample via a connectable feature. Figure 9Figure C illustrates an exemplary embodiment of the binding of a substitution probe to an amplification probe. In this example, binding is aided by a stalk feature that partially covers the amplification probe binding site A3+ on the amplification probe, which facilitates the binding of the substitution probe compared to the binding of R3- to R3+ on the primary probe. In this exemplary embodiment, the stalk consists of nucleotides 1 to n of the amplification probe binding site A3+, for example, with a total length of 10 nt. While the substitution probe in this exemplary embodiment can remain bound to the amplification probe, it allows the same amplification probe to be used for exponential amplification epochs when the substitution probe is not applied to the sample. Figure 9 Figure D illustrates an exemplary implementation where the replacement probe remains attached to the amplification probe after excess replacement probe is washed away. The replacement probe remains on the amplification probe to prevent the amplification probe from rebinding to the primary probe. Figure 9 Figure E illustrates an exemplary implementation of a subsequent amplification wheel, in which another amplification probe binds to the R3+ feature of the primary probe and binds to the sample via a connectable feature (circle).

[0026] Figure 10 An example design is provided for a scheme that iteratively accumulates probes near the primary probe binding site by using photocrosslinking, amplification probe cleavage, and subsequent washing to dissociate from the primary probe. Figure 10 Figure A illustrates exemplary implementations of primary probe binding to target molecules and amplification probe binding to primary probes, similar to... Figure 7 A. Figure 10 Figure B illustrates an exemplary implementation showing a more detailed view of the binding of the amplification probe to the primary probe at the R3+ / R3- site. The crossed circles in this example indicate that the amplification probe binds to the sample via a connectable feature. The white rectangles illustrate examples of the cleavable features of the amplification probe. Figure 10 Figure C illustrates an exemplary embodiment of an amplification probe cleaved at a cleavable feature. The portion of the amplification probe containing the binding feature A3+ remains near the primary probe because it is connected to the sample via the connectable feature indicated by the cross-circle. Figure 10 Figure D illustrates an exemplary implementation of the remaining portion of a cleaved amplification probe, which still binds to the example binding site R3+ via the binding feature R3-. This portion of the cleaved amplification probe can be removed, for example, by washing under conditions suitable for a short remaining fragment. Figure 10 Figure E illustrates an exemplary implementation where another amplification probe binds to the primary probe's feature R3+ and is bound to the sample via a connectable feature (circle), which is illustrated in the example. Figure 10 The steps described in BD become available.

[0027] Figure 11 An example image depicts the linear amplification of fluorescence signal intensity of a single gene in a cell culture sample using diaziridine-modified oligonucleotides. Figure 11 Figure A illustrates an exemplary implementation of increasing fluorescence intensity signal during multiple rounds of linear amplification. Figure 11 Figure B illustrates an exemplary implementation of an increase in fluorescence intensity signal after five rounds of continuous linear amplification. Figure 11 Figure C illustrates an exemplary implementation of an increase in fluorescence intensity signal after eleven rounds of continuous linear amplification.

[0028] Figure 12 An example image depicting the linear amplification of fluorescence signal intensity of a single gene in a cell culture sample using benzophenone-modified oligonucleotides. Scale bar: 20 micrometers. Figure 12 An exemplary embodiment illustrating the increase in fluorescence intensity signal during multiple rounds of linear amplification of benzophenone-functionalized oligonucleotides is shown.

[0029] Figure 13 The illustration shows an exemplary implementation of fluorescence intensity distribution changes during multiple rounds of linear amplification of benzophenone-functionalized oligonucleotides.

[0030] Figure 14 An exemplary embodiment of a linear and exponential amplification process using benzophenone-modified oligonucleotides to combine fluorescence signal intensities of a single gene in a cell culture sample is illustrated. Figure 14 Figure A illustrates an exemplary implementation of increased fluorescence intensity signal. The markers above the example image indicate the total number of amplification rounds. The contrast has been adjusted so that the images appear similar despite the increased fluorescence, to illustrate the similarity of fluorescence signal characteristics (such as spot size and number of spots) across different amplification rounds. Figure 14 Figure B illustrates an exemplary implementation of increased fluorescence intensity signal during multiple rounds of amplification of benzophenone-functionalized oligonucleotides. In this example image, the peak intensity of a single point is plotted for each imaging round, and the fold increase in fluorescence intensity is written above the corresponding data point.

[0031] Figure 15 The diagram illustrates an additional design for crosslinking amplification. Figure 15 Figure A illustrates a schematic diagram of the sacrificial layer design used for amplification. Figure 15 Diagram B illustrates the implementation. Figure 15 Scheme A shows the signal quantification of a single amplified point in a cell. The histogram shows the intensity of a single point in the cell compared to unamplified single-molecule FISH (smFISH) imaging. Figure 15 Figure C illustrates a bridging aptamer design that generates a signal only when the two amplicones are physically close to each other. The bridge probes, highlighted in bold, show the bridging aptamer binding across the two amplicones. The bold lines indicate the readout probe binding sites. Figure 15 Diagram D illustrates the use Figure 15Cell images amplified using the protocol shown in Figure C. Furthermore, non-specific amplification points in each amplification channel (three images on the left) were not observed in the bridging aptamer channel (image on the right). A magnified image shown within a white box is displayed in the lower right corner of each image. Arrows in the lower right inset indicate areas where non-specific points appear in a single amplification channel but not in the bridging channel.

[0032] Figure 16 The illustration shows an exemplary implementation of a linear and exponential amplification process using benzophenone-modified oligonucleotides to combine fluorescence signal intensities of a single gene in a cell culture sample. Figure 16 Illustration A illustrates an exemplary implementation of an increase in fluorescence intensity signal in a small portion of a recorded image to visualize a single fluorescence intensity peak. The markers above the example images indicate the total number of amplification epochs. All example images are contrast-matched, with the two rightmost images using a 1 / 10th short exposure time to illustrate the increase in signal intensity. Figure 16 Figure B illustrates an exemplary implementation of increased fluorescence intensity signal during multiple rounds of amplification of benzophenone-functionalized oligonucleotides. In this example image, the peak intensity of a single point is plotted for each imaging round, and the fold increase in fluorescence intensity is written above the corresponding data point.

[0033] Figure 17 The illustration shows that the probe can be cross-linked into cells via the CuAAC reaction. Figure 17 A is a schematic diagram of DNA oligonucleotides being "clicked" into the cell via 3' azide modification. Figure 17 B is a schematic diagram of the CuAAC crosslinking efficiency experiment. The primary probe and the amplification probe for testing are designed as shown in the figure; only the amplification probe is crosslinked into the cells. Figure 17 Figure C illustrates an example of smFISH signals from one round of crosslinking amplification probes on 24 Eef2 primary probes. Clicked and non-clicked signals, and before and after 60% formamide washing, are shown for comparison, with contrast adjusted to the same value for each channel. Figure 17 Figure D illustrates the quantization of the signal strength (peak value) in C. The image was Z-projected for quantization.

[0034] Figure 18 The illustration shows how split design can improve the targeting specificity of amplification probes. Figure 18 A is a schematic diagram of the probe click crosslinking amplification method. Figure 17 Figure B illustrates examples of Eef2 mRNA signaling before (right, A488) and after (left, Cy3B) 8 rounds of split probe amplification.

[0035] Figure 19 The diagram illustrates how branched designs can achieve rapid amplification using short amplification probes. Figure 19 A is a schematic diagram of the branched probe click crosslinking amplification method. Figure 19BC, an example of Eef2 signal after 6 rounds of amplification using 24 primary probes. Figure 19 Figure B illustrates that all amplification probe binding sites were designed to be 13 nucleotides (nt) (13nt BS). Figure 19 Figure C illustrates that all amplification probe binding sites were designed to be 15 nucleotides (15nt BS). Figure 19 Figure D illustrates the quantization of the dot peak intensity of standard smFISH, 13nt BS amplification, and 15nt BS amplification. The fold increases for 13nt BS were 19.35 (mean) and 16.64 (median); the fold increases for 15nt BS were 39.43 (mean) and 37.36 (median). Z-projection was performed on the images for quantization.

[0036] Figure 20 The illustration shows that the padlock design can reduce noise through exonuclease digestion. Figure 20 A is a schematic diagram of the padlock probe click crosslinking amplification method. Figure 20 B is an example of a single Eef2 primary probe signal after 14 rounds of amplification. Figure 20 Figure C illustrates the quantification of peak intensity amplification from standard smFISH (24 primary probes) versus amplification from a single primary probe. The mean fold increase is 8.41, and the median fold increase is 7.67. The primary probes are expected to have 80% binding efficiency, therefore the fold increase of a single probe is estimated to be approximately 150-fold. Detailed Implementation

[0037] The following description is intended to enable those skilled in the art to make and use the disclosed subject matter and incorporate it into applications. Various modifications and uses in different applications will be apparent to those skilled in the art, and the general principles defined herein are applicable to a wide range of implementations. Therefore, this disclosure is not intended to be limited to the presented implementations, but rather to be accorded the broadest scope consistent with the disclosed principles and novel features.

[0038] definition

[0039] As used herein, the terms “about” or “approximately” when referring to numbers generally include numbers that are 5%, 10%, 15%, or 20% greater or less in either direction of the number, unless otherwise stated or clearly indicated from the context (unless the number would be less than 0% or more than 100% of the possible value).

[0040] The term "oligonucleotide" refers to a polymer or oligomer of nucleotide monomers containing any combination of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges, or modified bridges.

[0041] Oligonucleotides can have various lengths. In certain embodiments, the length of an oligonucleotide can range from about 2 to about 500 nucleotides. In various related embodiments, the lengths of single-stranded, double-stranded, and triple-stranded oligonucleotides can range from about 12 to about 20 nucleotides, about 10 to about 60 nucleotides, about 10 to about 90 nucleotides, about 15 to about 30 nucleotides, and about 20 to about 120 nucleotides. In some embodiments, the length of an oligonucleotide is about 4 to about 39 nucleotides. In some embodiments, the length of an oligonucleotide is at least 4 nucleotides. In some embodiments, the length of an oligonucleotide is at least 5 nucleotides. In some embodiments, the length of an oligonucleotide is at least 6 nucleotides. In some embodiments, the length of an oligonucleotide is at least 7 nucleotides. In some embodiments, the length of an oligonucleotide is at least 8 nucleotides. In some embodiments, the length of an oligonucleotide is at least 9 nucleotides. In some embodiments, the length of an oligonucleotide is at least 10 nucleotides. In some embodiments, the length of an oligonucleotide is at least 11 nucleotides. In some embodiments, the length of an oligonucleotide is at least 12 nucleotides. In some embodiments, the length of an oligonucleotide is at least 15 nucleotides. In some embodiments, the oligonucleotide is at least 20 nucleotides long. In some embodiments, the oligonucleotide is at least 25 nucleotides long. In some embodiments, the oligonucleotide is at least 30 nucleotides long. In some embodiments, the oligonucleotide is a duplex of complementary strands at least 18 nucleotides long. In some embodiments, the oligonucleotide is a duplex of complementary strands at least 21 nucleotides long.

[0042] As used herein, the term "probe" refers to any synthetic or naturally occurring molecule that can be directly or indirectly attached to a molecular target (e.g., an mRNA sample, DNA molecule, protein molecule, RNA and DNA subtype molecules, single nucleotide polymorphism molecules, etc.). For example, a probe may include nucleic acid molecules, oligonucleotides, proteins (e.g., antibody or antigen-binding sequences), or combinations thereof. For example, a protein probe may link one or more nucleic acid molecules to form a chimeric probe. As disclosed herein, in some embodiments, the probe itself may generate a detectable signal. In some embodiments, the probe is directly or indirectly linked via an intermediate molecule to a signal portion (e.g., a dye or fluorophore) that can generate a detectable signal.

[0043] As used herein, the term "sample" refers to a biological sample obtained or derived from a source of interest, as described herein. In some embodiments, the source of interest includes organisms such as animals, plants, microorganisms, or humans. In some embodiments, the biological sample includes biological tissues or body fluids. In some embodiments, the biological sample is or includes bone marrow; blood; blood cells; ascites; tissue or fine-needle biopsy samples; cellular body fluids; cell-containing nucleic acids; sputum; saliva; urine; cerebrospinal fluid; peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washing or lavage solutions, such as catheter lavage or bronchoalveolar lavage fluid; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces; other body fluids, secretions, and / or excretions; and / or cells derived therefrom. In some embodiments, the biological sample is or includes cells obtained from an individual. In some embodiments, the sample is a "primary sample" obtained directly from the source of interest by any suitable means. For example, in some embodiments, the primary biological sample is obtained by methods selected from: biopsy (e.g., fine-needle aspiration or tissue biopsy), surgery, body fluid collection (e.g., blood, lymph, feces, etc.). In some embodiments, as the context clearly indicates, the term "sample" refers to a preparation obtained by processing the primary sample (e.g., by removing one or more components of the primary sample and / or by adding one or more reagents to the primary sample). For example, semi-permeable membrane filtration is used. Such a "processed sample" may contain, for example, nucleic acids or proteins extracted from the sample, or obtained by subjecting the primary sample to techniques such as mRNA amplification or reverse transcription, separation and / or purification of certain components. In some embodiments, the term "sample" refers to nucleic acids, such as DNA, RNA, transcripts, or chromosomes. In some embodiments, the term "sample" refers to nucleic acids extracted from cells.

[0044] As used herein, the terms “target analyte” or “analyte” refer to transcripts, RNA, DNA sites, chromosomes, DNA, exogenous DNA, proteins, peptides, lipids, polysaccharides, cellular components, small molecules, metabolites, primary probes, amplification probes, organelles, and any combination thereof.

[0045] As used herein, the term "cellular component" refers to structures in a cell that regulate movement, maintain cell shape, produce proteins, or any combination thereof. In some embodiments, cellular components are selected from the nucleolus, nucleus, ribosomes, vesicles, rough endoplasmic reticulum, Golgi apparatus, cytoskeleton, smooth endoplasmic reticulum, mitochondria, vacuoles, cytoplasm, lysosomes, centrosomes, cell membrane, and any combination thereof. In some embodiments, "cellular component" refers to transcripts, RNA, DNA sites, chromosomes, DNA, proteins, peptides, lipids, glycans, small molecules, metabolites, primary probes, amplification probes, organelles, and any combination thereof.

[0046] As used herein, the term "primary probe" refers to a probe that interacts with a target analyte. In some examples, the probe is a nucleic acid molecule. In some examples, the probe is an oligonucleotide.

[0047] As used herein, the term "amplification probe" refers to a probe that interacts with a primary probe or another amplification probe. A "primary amplification" probe is an amplification probe that interacts with a primary probe. A "secondary amplification probe" is an amplification probe that interacts with or with a primary amplification probe. A "tertiary amplification probe" is an amplification probe that interacts with a secondary amplification probe or with a primary amplification probe. A "quaternary amplification probe" is an amplification probe that interacts with a tertiary amplification probe or with a secondary amplification probe. In some embodiments, the amplification probe is a nucleic acid sequence. In some embodiments, the amplification probe is an oligonucleotide. In some embodiments, the amplification probe has a motif cross-linked to cellular components.

[0048] As used herein, the term "readout probe" refers to a probe that interacts with an amplification probe. In some embodiments, the readout probe is a nucleic acid sequence containing one or more fluorophores. In some embodiments, the readout probe is an oligonucleotide.

[0049] As disclosed herein, the term "barcode" refers to a sequence signal generated by the method described herein. Barcode sequences are typically long and unique enough to identify molecular targets in a single cell.

[0050] As disclosed herein, the term "cellular component" refers to a target selected from transcripts, RNA, DNA sites, chromosomes, DNA, proteins, antibodies, lipids, glycans, cellular components, organelles, synapses, intercellular connections, cellular component boundaries, and any combination thereof.

[0051] As disclosed herein, the term "distribution" refers to the location of a cellular component within a cell. In some embodiments, the term "distribution" also refers to the interactions between cellular components and between other cellular components.

[0052] As disclosed herein, the term “map” refers to detecting barcodes linked to cellular components and identifying their location inside or outside the cell.

[0053] As disclosed herein, the term "interaction" refers to the binding of two or more molecules. In some embodiments, the term "interaction" is synonymous with "binding." In some embodiments, binding may occur through intermolecular forces between two or more molecules, such as ionic bonds, hydrogen bonds, and van der Waals forces. In some embodiments, binding may refer to the formation of a covalent bond between two or more molecules. In some embodiments, "binding" may refer to the direct or indirect interaction of two or more molecules. In some embodiments, two or more molecules that are indirectly bound have one or more molecules that interact between them. For example, if molecule C interacts with molecules A and B, but molecules A and B do not interact directly, then molecules A and B interact indirectly. In some embodiments, "binding" may refer to the hybridization of two or more nucleotide sequences. In some embodiments, "binding" may refer to protein-nucleotide interactions. In some embodiments, "binding" may refer to protein-protein interactions.

[0054] As defined in this article, the term “near distance” refers to the distance between two objects, where the first object is approximately 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, or 3000 nanometers from the other object.

[0055] Overview

[0056] This disclosure provides a method for scalably amplifying analyte signals in a sample to spatially locate the analyte in a biological sample. The disclosure describes the method, as well as solutions to problems using these methods and in other related fields.

[0057] In some embodiments, a method is provided for scalably amplifying an analyte signal in a sample to spatially localize the analyte in a biological sample, comprising: contacting one or more target analytes in the sample with a plurality of primary probes, wherein each of the plurality of primary probes interacts with at least one target analyte. In some embodiments, the method comprises: contacting each of the plurality of primary probes with one or more amplification probes, each primary probe interacting with at least one analyte. In some embodiments, the method comprises: optionally, crosslinking one or more amplification probes to a cellular component. In some embodiments, the method comprises: optionally, separating each of the one or more amplification probes from its primary probe or from another amplification probe. In some embodiments, the method comprises: optionally, repeating any of the foregoing embodiments individually or in combination. In some embodiments, the method comprises: detecting one or more target analytes.

[0058] In some embodiments, the method further includes contacting one or more amplification probes with one or more readout probes.

[0059] In some implementations, the method includes detecting the signal by imaging or sequencing. In some implementations, one or more target analytes are detected by signal or signal absence.

[0060] In some embodiments, the method includes at least one step of crosslinking one or more amplification probes to a cellular component.

[0061] In some implementations, the method includes at least one step of separating each of one or more amplification probes from its primary probe or from another amplification probe.

[0062] In some embodiments, the method further includes contacting one or more amplification probes with another amplification probe.

[0063] In some implementations, the method includes imaging the sample after contacting the amplification probe with one or more readout probes.

[0064] In some embodiments, the method further includes amplifying the amplification probe by contacting the amplification probe from a previous contact step with a plurality of new amplification probes.

[0065] In some embodiments, the method further includes: repeating the steps of contacting the amplification probe with one or more readout probes and the imaging step, each time using a new plurality of readout probes, such that the target analyte is described by a barcode and can be distinguished from another target analyte in the sample by the difference in its barcode. In some embodiments, the method includes: optionally, separating the amplification probe from the primary probe or another amplification probe after imaging the sample. In some embodiments, the method includes: optionally, separating the readout probe from the amplification probe.

[0066] In some embodiments, the method includes single-molecule resolution of the target analyte. In some embodiments, the method includes single-molecule resolution of cellular components.

[0067] Target analytes and cellular components

[0068] In some embodiments, the target analyte includes transcripts, RNA, DNA sites, chromosomes, DNA, proteins, peptides, lipids, glycans, small molecules, metabolites, primary probes, amplification probes, organelles, membranes, and any combination thereof. In some embodiments, the target analyte is obtained from bacterial cells, archaea cells, eukaryotic cells, or combinations thereof.

[0069] In some embodiments, the target analyte includes a molecular target selected from proteins, modified proteins, transcripts, RNA, DNA sites, exogenous proteins, exogenous nucleic acids, hormones, carbohydrates, small molecules, bioactive molecules, and combinations thereof. In some embodiments, the target includes subcellular features. In some embodiments, the target analyte includes RNA-DNA interactions, RNA-protein interactions, DNA-protein interactions, protein-protein interactions, or nucleic acid-small molecule interactions.

[0070] In some embodiments, the cellular component includes structures that regulate cell movement, maintain cell shape, produce proteins, or any combination thereof. In some embodiments, the cellular component is selected from the nucleolus, nucleus, ribosome, vesicle, rough endoplasmic reticulum, Golgi apparatus, cytoskeleton, smooth endoplasmic reticulum, mitochondria, vacuoles, cytoplasm, lysosomes, centrosomes, cell membrane, and any combination thereof.

[0071] In some embodiments, the cellular components include transcripts, RNA, DNA sites, chromosomes, DNA, proteins, peptides, lipids, polysaccharides, small molecules, metabolites, primary probes, amplification probes, organelles, and any combination thereof.

[0072] In some embodiments, the cellular component is a target analyte. In some embodiments, the cellular component and the target analyte are the same. In some embodiments, the cellular component and the target analyte are different. In some embodiments, the cellular component is located approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 nanometers from the target analyte.

[0073] Primary probe

[0074] In some embodiments, the primary probe is selected from proteins, modified proteins, RNA, oligonucleotides, antibodies, antibody fragments, and combinations thereof. In some embodiments, the primary probe comprises an oligonucleotide.

[0075] In some embodiments, the primary probe comprises an oligonucleotide with a length of at least 5 nucleotides. In some embodiments, the primary probe comprises an oligonucleotide with a length of at least 6 nucleotides. In some embodiments, the primary probe comprises an oligonucleotide with a length of at least 7 nucleotides. In some embodiments, the primary probe comprises an oligonucleotide with a length of at least 8 nucleotides. In some embodiments, the primary probe comprises an oligonucleotide with a length of at least 9 nucleotides. In some embodiments, the primary probe comprises an oligonucleotide with a length of at least 10 nucleotides. In some embodiments, the primary probe comprises an oligonucleotide with a length of at least 11 nucleotides. In some embodiments, the primary probe comprises an oligonucleotide with a length of at least 12 nucleotides. In some embodiments, the primary probe comprises an oligonucleotide with a length of at least 13 nucleotides. In some embodiments, the primary probe comprises an oligonucleotide with a length of at least 14 nucleotides. In some embodiments, the primary probe comprises an oligonucleotide with a length of at least 15 nucleotides. In some embodiments, the primary probe comprises an oligonucleotide with a length of at least 16 nucleotides. In some embodiments, the primary probe comprises an oligonucleotide with a length of at least 17 nucleotides. In some embodiments, the primary probe comprises an oligonucleotide with a length of at least 18 nucleotides. In some embodiments, the primary probe comprises an oligonucleotide with a length of at least 19 nucleotides. In some embodiments, the primary probe comprises an oligonucleotide with a length of at least 20 nucleotides. In some embodiments, the primary probe comprises an oligonucleotide with a length of at least 21 nucleotides. In some embodiments, the primary probe comprises an oligonucleotide with a length of at least 22 nucleotides. In some embodiments, the primary probe comprises an oligonucleotide with a length of at least 23 nucleotides. In some embodiments, the primary probe comprises an oligonucleotide with a length of at least 24 nucleotides. In some embodiments, the primary probe comprises an oligonucleotide with a length of at least 25 nucleotides. In some embodiments, the primary probe comprises an oligonucleotide with a length of at least 26 nucleotides. In some embodiments, the primary probe comprises an oligonucleotide with a length of at least 27 nucleotides. In some embodiments, the primary probe comprises an oligonucleotide with a length of at least 28 nucleotides. In some embodiments, the primary probe comprises an oligonucleotide with a length of at least 29 nucleotides. In some embodiments, the primary probe comprises an oligonucleotide with a length of at least 30 nucleotides. In some embodiments, the primary probe of any of the foregoing embodiments comprises an oligonucleotide with a length of less than 35, 40, 45, 50, 100, 150, 200, 250 or 300 nucleotides.

[0076] In some embodiments, the primary probe selectively interacts with the target analyte. In some embodiments, the primary probe is an antibody that interacts with the target analyte. In some embodiments, the primary probe is an antibody-oligonucleotide conjugate, wherein the oligonucleotide interacts with the target analyte.

[0077] In some embodiments, the primary probe comprises a sequence complementary to the target analyte. In some embodiments, the sequence complementarity comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0078] In some embodiments, the primary probe includes one or more amplification probe binding sites. In some embodiments, the primary probe includes two or more amplification probe binding sites. In some embodiments, the primary probe includes three or more amplification probe binding sites. In some embodiments, the primary probe includes four or more amplification probe binding sites. In some embodiments, the primary probe includes five or more amplification probe binding sites. In some embodiments, the primary probe includes six or more amplification probe binding sites. In some embodiments, the primary probe includes seven or more amplification probe binding sites. In some embodiments, the primary probe includes eight or more amplification probe binding sites.

[0079] In some embodiments, the one or more amplification probe binding sites are the same sequence. In some embodiments, at least one amplification probe binding site in the one or more amplification probe sequences is the same. In some embodiments, the one or more amplification probe binding sites are different from each other.

[0080] In some embodiments, the amplification probe binding site comprises a nucleotide sequence of at least 5 nucleotides in length. In some embodiments, the amplification probe binding site comprises a nucleotide sequence of at least 6 nucleotides in length. In some embodiments, the amplification probe binding site comprises a nucleotide sequence of at least 7 nucleotides in length. In some embodiments, the amplification probe binding site comprises a nucleotide sequence of at least 8 nucleotides in length. In some embodiments, the amplification probe binding site comprises a nucleotide sequence of at least 9 nucleotides in length. In some embodiments, the amplification probe binding site comprises a nucleotide sequence of at least 10 nucleotides in length. In some embodiments, the amplification probe binding site comprises a nucleotide sequence of at least 11 nucleotides in length. In some embodiments, the amplification probe binding site comprises a nucleotide sequence of at least 12 nucleotides in length. In some embodiments, the amplification probe binding site comprises a nucleotide sequence of at least 13 nucleotides in length. In some embodiments, the amplification probe binding site comprises a nucleotide sequence of at least 14 nucleotides in length. In some embodiments, the amplification probe binding site comprises a nucleotide sequence of at least 15 nucleotides in length. In some embodiments, the amplification probe binding site comprises a nucleotide sequence of at least 16 nucleotides in length. In some embodiments, the amplification probe binding site comprises a nucleotide sequence of at least 17 nucleotides in length. In some embodiments, the amplification probe binding site comprises a nucleotide sequence of at least 18 nucleotides in length. In some embodiments, the amplification probe binding site comprises a nucleotide sequence of at least 19 nucleotides in length. In some embodiments, the amplification probe binding site comprises a nucleotide sequence of at least 20 nucleotides in length. In some embodiments, the amplification probe binding site comprises a nucleotide sequence of at least 21 nucleotides in length. In some embodiments, the amplification probe binding site comprises a nucleotide sequence of at least 22 nucleotides in length. In some embodiments, the amplification probe binding site comprises a nucleotide sequence of at least 23 nucleotides in length. In some embodiments, the amplification probe binding site comprises a nucleotide sequence of at least 24 nucleotides in length. In some embodiments, the amplification probe binding site comprises a nucleotide sequence of at least 25 nucleotides in length. In some embodiments, the amplification probe binding site comprises a nucleotide sequence of at least 26 nucleotides in length. In some embodiments, the amplification probe binding site comprises a nucleotide sequence of at least 27 nucleotides in length. In some embodiments, the amplification probe binding site comprises a nucleotide sequence of less than 35, 40, 45, 50, 100, 150, 200, 250, or 300 nucleotides in length.

[0081] In some embodiments, the primary probe includes one or more analyte binding sites. In some embodiments, the analyte binding sites on the primary probe are identical. In some embodiments, the analyte binding sites on the primary probe are different. In some embodiments, the analyte binding site includes a sequence complementary to the target analyte. In some embodiments, the sequence complementarity includes at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0082] In some embodiments, the analyte binding site comprises a nucleotide sequence of at least 5 nucleotides in length. In some embodiments, the analyte binding site comprises a nucleotide sequence of at least 6 nucleotides in length. In some embodiments, the analyte binding site comprises a nucleotide sequence of at least 7 nucleotides in length. In some embodiments, the analyte binding site comprises a nucleotide sequence of at least 8 nucleotides in length. In some embodiments, the analyte binding site comprises a nucleotide sequence of at least 9 nucleotides in length. In some embodiments, the analyte binding site comprises a nucleotide sequence of at least 10 nucleotides in length. In some embodiments, the analyte binding site comprises a nucleotide sequence of at least 11 nucleotides in length. In some embodiments, the analyte binding site comprises a nucleotide sequence of at least 12 nucleotides in length. In some embodiments, the analyte binding site comprises a nucleotide sequence of at least 13 nucleotides in length. In some embodiments, the analyte binding site comprises a nucleotide sequence of at least 14 nucleotides in length. In some embodiments, the analyte binding site comprises a nucleotide sequence of at least 15 nucleotides in length. In some embodiments, the analyte binding site comprises a nucleotide sequence of at least 16 nucleotides in length. In some embodiments, the analyte binding site comprises a nucleotide sequence of at least 17 nucleotides in length. In some embodiments, the analyte binding site comprises a nucleotide sequence of at least 18 nucleotides in length. In some embodiments, the analyte binding site comprises a nucleotide sequence of at least 19 nucleotides in length. In some embodiments, the analyte binding site comprises a nucleotide sequence of at least 20 nucleotides in length. In some embodiments, the analyte binding site comprises a nucleotide sequence of at least 21 nucleotides in length. In some embodiments, the analyte binding site comprises a nucleotide sequence of at least 22 nucleotides in length. In some embodiments, the analyte binding site comprises a nucleotide sequence of at least 23 nucleotides in length. In some embodiments, the analyte binding site comprises a nucleotide sequence of at least 24 nucleotides in length. In some embodiments, the analyte binding site comprises a nucleotide sequence of at least 25 nucleotides in length. In some embodiments, the analyte binding site comprises a nucleotide sequence of at least 26 nucleotides in length. In some embodiments, the analyte binding site comprises a nucleotide sequence of at least 27 nucleotides in length. In some embodiments, the analyte binding site comprises a nucleotide sequence of less than 35, 40, 45, 50, 100, 150, 200, 250, or 300 nucleotides in length.

[0083] Amplification probe

[0084] In some embodiments, the amplification probe is a nucleic acid sequence that interacts with a primary probe or another amplification probe. In some embodiments, the amplification probe is an oligonucleotide. In some embodiments, one or more amplification probes include secondary, tertiary, and quaternary amplification probes.

[0085] In some embodiments, the amplification probe interacts with a primary probe, which is a protein, through interaction with a nucleotide binding site on the protein. In some embodiments, the amplification probe interacts with a primary probe, which is an antibody. In some embodiments, the amplification probe interacts with a primary probe, which is a protein-oligonucleotide conjugate, through interaction with an oligonucleotide bound to the conjugate. In some embodiments, the amplification probe interacts with a primary probe, which is an antibody-oligonucleotide conjugate, through interaction with an oligonucleotide bound to the conjugate.

[0086] In some embodiments, the amplification probe includes one or more primary probe binding sites. In some embodiments, one or more amplification probes bind to a contact site on at least one primary probe. In some embodiments, one or more amplification probes include binding sites that are inversely complementary to the binding sites of one or more primary probes. In some embodiments, one or more amplification probes include one or more unique readout sites, or repeat sequences of the same readout site on a primary probe or another amplification probe.

[0087] In some embodiments, the amplification probe includes a primary probe binding site, a portion allowing the amplification probe to dissociate from the primary probe, one or more identical amplification probe binding sites, one or more different amplification probe binding sites, one or more identical readout probe binding sites, one or more different readout probe binding sites, one or more different crosslinking sites, a secondary probe binding site, a tertiary probe binding site, or any combination thereof. In some embodiments, the secondary probe binding site includes a nucleotide sequence that binds to another amplification probe. In some embodiments, the tertiary probe binding site includes a nucleotide sequence that binds to another amplification probe. In some embodiments, the primary probe includes one or more primary probe binding sites. In some embodiments, the primary probe includes one or more portions allowing the amplification probe to dissociate from the primary probe. In some embodiments, the primary probe includes one or more identical amplification probe binding sites. In some embodiments, the primary probe includes one or more different amplification probe binding sites. In some embodiments, the primary probe includes one or more identical readout probe binding sites. In some embodiments, the primary probe includes one or more different readout probe binding sites. In some embodiments, the primary probe includes one or more different crosslinking sites. In some embodiments, the primary probe includes one or more secondary probe binding sites. In some implementations, the primary probe includes one or more tertiary probe binding sites.

[0088] In some embodiments, the amplification probe is at least 5 nucleotides long. In some embodiments, the amplification probe is at least 6 nucleotides long. In some embodiments, the amplification probe is at least 7 nucleotides long. In some embodiments, the amplification probe is at least 8 nucleotides long. In some embodiments, the amplification probe is at least 9 nucleotides long. In some embodiments, the amplification probe is at least 10 nucleotides long. In some embodiments, the amplification probe is at least 11 nucleotides long. In some embodiments, the amplification probe is at least 12 nucleotides long. In some embodiments, the amplification probe is at least 13 nucleotides long. In some embodiments, the amplification probe is at least 14 nucleotides long. In some embodiments, the amplification probe is at least 15 nucleotides long. In some embodiments, the amplification probe is at least 16 nucleotides long. In some embodiments, the amplification probe is at least 17 nucleotides long. In some embodiments, the amplification probe is at least 18 nucleotides long. In some embodiments, the amplification probe is at least 19 nucleotides long. In some embodiments, the amplification probe is at least 20 nucleotides long. In some embodiments, the amplification probe is at least 21 nucleotides long. In some embodiments, the amplification probe is at least 22 nucleotides long. In some embodiments, the amplification probe is at least 23 nucleotides long. In some embodiments, the amplification probe is at least 24 nucleotides long. In some embodiments, the amplification probe is at least 25 nucleotides long. In some embodiments, the amplification probe is at least 26 nucleotides long. In some embodiments, the amplification probe is at least 27 nucleotides long. In some embodiments, the amplification probe is at least 28 nucleotides long. In some embodiments, the amplification probe is at least 29 nucleotides long. In some embodiments, the amplification probe is at least 30 nucleotides long. In some embodiments, the amplification probe of any of the foregoing embodiments comprises a nucleotide sequence less than 35, 40, 45, 50, 100, 150, 200, 250, or 300 nucleotides in length.

[0089] In some embodiments, the amplification probe includes a primary probe binding site. In some embodiments, the primary probe binding site interacts with the primary probe.

[0090] In some embodiments, the primary probe binding site comprises a sequence complementary to the primary probe. In some embodiments, the sequence complementarity comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0091] In some embodiments, the primary probe binding site comprises a nucleotide sequence of at least 5 nucleotides in length. In some embodiments, the primary probe binding site comprises a nucleotide sequence of at least 6 nucleotides in length. In some embodiments, the primary probe binding site comprises a nucleotide sequence of at least 7 nucleotides in length. In some embodiments, the primary probe binding site comprises a nucleotide sequence of at least 8 nucleotides in length. In some embodiments, the primary probe binding site comprises a nucleotide sequence of at least 9 nucleotides in length. In some embodiments, the primary probe binding site comprises a nucleotide sequence of at least 10 nucleotides in length. In some embodiments, the primary probe binding site comprises a nucleotide sequence of at least 11 nucleotides in length. In some embodiments, the primary probe binding site comprises a nucleotide sequence of at least 12 nucleotides in length. In some embodiments, the primary probe binding site comprises a nucleotide sequence of at least 13 nucleotides in length. In some embodiments, the primary probe binding site comprises a nucleotide sequence of at least 14 nucleotides in length. In some embodiments, the primary probe binding site comprises a nucleotide sequence of at least 15 nucleotides in length. In some embodiments, the primary probe binding site comprises a nucleotide sequence of at least 16 nucleotides in length. In some embodiments, the primary probe binding site comprises a nucleotide sequence of at least 17 nucleotides in length. In some embodiments, the primary probe binding site comprises a nucleotide sequence of at least 18 nucleotides in length. In some embodiments, the primary probe binding site comprises a nucleotide sequence of at least 19 nucleotides in length. In some embodiments, the primary probe binding site comprises a nucleotide sequence of at least 20 nucleotides in length. In some embodiments, the primary probe binding site comprises a nucleotide sequence of at least 21 nucleotides in length. In some embodiments, the primary probe binding site comprises a nucleotide sequence of at least 22 nucleotides in length. In some embodiments, the primary probe binding site comprises a nucleotide sequence of at least 23 nucleotides in length. In some embodiments, the primary probe binding site comprises a nucleotide sequence of at least 24 nucleotides in length. In some embodiments, the primary probe binding site comprises a nucleotide sequence of at least 25 nucleotides in length. In some embodiments, the primary probe binding site comprises a nucleotide sequence of at least 26 nucleotides in length. In some embodiments, the primary probe binding site comprises a nucleotide sequence of at least 27 nucleotides in length. In some embodiments, the primary probe binding site comprises a nucleotide sequence of less than 35, 40, 45, 50, 100, 150, 200, 250, or 300 nucleotides in length.

[0092] In some embodiments, the amplification probe includes a portion that allows the amplification probe to dissociate from the primary probe. In some embodiments, the dissociation is a disruption of the interaction between the amplification probe and the primary probe. In some embodiments, the amplification probe includes a portion that allows the amplification probe to dissociate from the readout probe. In some embodiments, the dissociation is a disruption of the interaction between the amplification probe and the readout probe.

[0093] In some embodiments, this portion is a substitution probe binding site. In some embodiments, this portion contains a sequence complementary to the primary probe or amplification probe. In some embodiments, the sequence complementarity comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0094] In some embodiments, the portion comprises a nucleotide sequence of at least 5 nucleotides in length. In some embodiments, the portion comprises a nucleotide sequence of at least 6 nucleotides in length. In some embodiments, the portion comprises a nucleotide sequence of at least 7 nucleotides in length. In some embodiments, the portion comprises a nucleotide sequence of at least 8 nucleotides in length. In some embodiments, the portion comprises a nucleotide sequence of at least 9 nucleotides in length. In some embodiments, the portion comprises a nucleotide sequence of at least 10 nucleotides in length. In some embodiments, the portion comprises a nucleotide sequence of at least 11 nucleotides in length. In some embodiments, the portion comprises a nucleotide sequence of at least 12 nucleotides in length. In some embodiments, the portion comprises a nucleotide sequence of at least 13 nucleotides in length. In some embodiments, the portion comprises a nucleotide sequence of at least 14 nucleotides in length. In some embodiments, the portion comprises a nucleotide sequence of at least 15 nucleotides in length. In some embodiments, the portion comprises a nucleotide sequence of at least 16 nucleotides in length. In some embodiments, the portion comprises a nucleotide sequence of at least 17 nucleotides in length. In some embodiments, the portion comprises a nucleotide sequence of at least 18 nucleotides in length. In some embodiments, the portion comprises a nucleotide sequence of at least 19 nucleotides in length. In some embodiments, the portion comprises a nucleotide sequence of at least 20 nucleotides in length. In some embodiments, the portion comprises a nucleotide sequence of at least 21 nucleotides in length. In some embodiments, the portion comprises a nucleotide sequence of at least 22 nucleotides in length. In some embodiments, the portion comprises a nucleotide sequence of at least 23 nucleotides in length. In some embodiments, the portion comprises a nucleotide sequence of at least 24 nucleotides in length. In some embodiments, the portion comprises a nucleotide sequence of at least 25 nucleotides in length. In some embodiments, the portion comprises a nucleotide sequence of at least 26 nucleotides in length. In some embodiments, the portion comprises a nucleotide sequence of at least 27 nucleotides in length. In some embodiments, the portion comprises a nucleotide sequence of less than 35, 40, 45, 50, 100, 150, 200, 250, or 300 nucleotides in length.

[0095] In some embodiments, the amplification probe includes one or more readout probe binding sites. In some embodiments, the readout probe includes a sequence complementary to the amplification probe. In some embodiments, the sequence complementarity includes at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0096] In some embodiments, the readout probe comprises a nucleotide sequence of at least 5 nucleotides in length. In some embodiments, the readout probe comprises a nucleotide sequence of at least 6 nucleotides in length. In some embodiments, the readout probe comprises a nucleotide sequence of at least 7 nucleotides in length. In some embodiments, the readout probe comprises a nucleotide sequence of at least 8 nucleotides in length. In some embodiments, the readout probe comprises a nucleotide sequence of at least 9 nucleotides in length. In some embodiments, the readout probe comprises a nucleotide sequence of at least 10 nucleotides in length. In some embodiments, the readout probe comprises a nucleotide sequence of at least 11 nucleotides in length. In some embodiments, the readout probe comprises a nucleotide sequence of at least 12 nucleotides in length. In some embodiments, the portion comprises a nucleotide sequence of at least 13 nucleotides in length. In some embodiments, the readout probe comprises a nucleotide sequence of at least 14 nucleotides in length. In some embodiments, the readout probe comprises a nucleotide sequence of at least 15 nucleotides in length. In some embodiments, the readout probe comprises a nucleotide sequence of at least 16 nucleotides in length. In some embodiments, the readout probe comprises a nucleotide sequence of at least 17 nucleotides in length. In some embodiments, the readout probe comprises a nucleotide sequence of at least 18 nucleotides in length. In some embodiments, the readout probe comprises a nucleotide sequence of at least 19 nucleotides in length. In some embodiments, the readout probe comprises a nucleotide sequence of at least 20 nucleotides in length. In some embodiments, the readout probe comprises a nucleotide sequence of at least 21 nucleotides in length. In some embodiments, the readout probe comprises a nucleotide sequence of at least 22 nucleotides in length. In some embodiments, the readout probe comprises a nucleotide sequence of at least 23 nucleotides in length. In some embodiments, the readout probe comprises a nucleotide sequence of at least 24 nucleotides in length. In some embodiments, the readout probe comprises a nucleotide sequence of at least 25 nucleotides in length. In some embodiments, the readout probe comprises a nucleotide sequence of at least 26 nucleotides in length. In some embodiments, the readout probe comprises a nucleotide sequence of at least 27 nucleotides in length. In some embodiments, the readout probe comprises a nucleotide sequence of less than 35, 40, 45, 50, 100, 150, 200, 250, or 300 nucleotides.

[0097] In some embodiments, the amplification probe includes one or more identical readout probe binding sites. In some embodiments, the identical readout probe binds to different locations on the amplification probe at the same readout probe binding site. In some embodiments, the amplification probe includes one or more different readout probe binding sites. In some embodiments, different readout probes bind to different locations on the amplification probe at different readout probe binding sites.

[0098] In some embodiments, the amplification probe includes one or more distinct cross-linking sites. In some embodiments, the cross-linking sites comprise regions of the amplification probe that can cross-link to cellular components.

[0099] In some implementations, one or more amplification probes bind to other amplification probes.

[0100] In some embodiments, the amplification probe includes a secondary probe binding site. In some embodiments, the secondary probe binding site includes a region that interacts with another amplification probe, thereby linking the primary amplification probe to the secondary amplification probe.

[0101] In some embodiments, the amplification probe includes a tertiary probe binding site. In some embodiments, the tertiary probe binding site includes a region that interacts with another amplification probe, thereby linking a primary amplification probe that interacts with a secondary amplification probe to the tertiary amplification probe.

[0102] In some embodiments, the amplification probe includes a quaternary probe binding site. In some embodiments, the quaternary probe binding site includes a region that interacts with another amplification probe, thereby linking a primary amplification probe interacting with a secondary amplification probe, a secondary amplification probe interacting with a tertiary probe, to the quaternary amplification probe.

[0103] In some implementations, the tertiary amplification probe interacts with the secondary amplification probe, and the secondary amplification probe interacts with the primary probe.

[0104] In some implementations, the quaternary amplification probe interacts with the tertiary amplification probe, the tertiary probe interacts with the secondary amplification probe, and the secondary probe interacts with the primary probe.

[0105] In some implementations, the quaternary amplification probe is the same as the 2nd-level amplification probe.

[0106] In some implementations, the quaternary amplification probe interacts with the secondary amplification probe.

[0107] In some embodiments, the quaternary amplification probe includes one or more binding sites for a secondary amplification probe, one or more binding sites for a tertiary amplification probe, or any combination thereof. In some embodiments, the tertiary amplification probe includes one or more binding sites for a secondary amplification probe, one or more binding sites for a quaternary amplification probe, or any combination thereof. In some embodiments, the secondary amplification probe includes one or more binding sites for a tertiary amplification probe, one or more binding sites for a quaternary amplification probe, one or more binding sites for a primary probe, or any combination thereof.

[0108] In some embodiments, the secondary and tertiary probe binding sites comprise sequences complementary to the primary amplification probe. In some embodiments, the sequence complementarity comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0109] In some embodiments, each of the secondary, tertiary, or quaternary amplification probes comprises a nucleotide sequence of at least 5 nucleotides in length. In some embodiments, each of the secondary, tertiary, or quaternary amplification probes comprises a nucleotide sequence of at least 6 nucleotides in length. In some embodiments, each of the secondary, tertiary, or quaternary amplification probes comprises a nucleotide sequence of at least 7 nucleotides in length. In some embodiments, each of the secondary, tertiary, or quaternary amplification probes comprises a nucleotide sequence of at least 8 nucleotides in length. In some embodiments, each of the secondary, tertiary, or quaternary amplification probes comprises a nucleotide sequence of at least 9 nucleotides in length. In some embodiments, each of the secondary, tertiary, or quaternary amplification probes comprises a nucleotide sequence of at least 10 nucleotides in length. In some embodiments, each of the secondary, tertiary, or quaternary amplification probes comprises a nucleotide sequence of at least 11 nucleotides in length. In some embodiments, each of the secondary, tertiary, or quaternary amplification probes comprises a nucleotide sequence of at least 12 nucleotides in length. In some embodiments, each of the tertiary or quaternary amplification probes comprises a nucleotide sequence of at least 13 nucleotides in length. In some embodiments, each of the secondary, tertiary, or quaternary amplification probes comprises a nucleotide sequence of at least 14 nucleotides in length. In some embodiments, each of the secondary, tertiary, or quaternary amplification probes comprises a nucleotide sequence of at least 15 nucleotides in length. In some embodiments, each of the secondary, tertiary, or quaternary amplification probes comprises a nucleotide sequence of at least 16 nucleotides in length. In some embodiments, each of the secondary, tertiary, or quaternary amplification probes comprises a nucleotide sequence of at least 17 nucleotides in length. In some embodiments, each of the secondary, tertiary, or quaternary amplification probes comprises a nucleotide sequence of at least 18 nucleotides in length. In some embodiments, each of the secondary, tertiary, or quaternary amplification probes comprises a nucleotide sequence of at least 19 nucleotides in length. In some embodiments, each of the secondary, tertiary, or quaternary amplification probes comprises a nucleotide sequence of at least 20 nucleotides in length. In some embodiments, each of the secondary, tertiary, or quaternary amplification probes comprises a nucleotide sequence of at least 21 nucleotides in length. In some embodiments, each of the secondary, tertiary, or quaternary amplification probes comprises a nucleotide sequence of at least 22 nucleotides in length. In some embodiments, each of the secondary, tertiary, or quaternary amplification probes comprises a nucleotide sequence of at least 23 nucleotides in length. In some embodiments, each of the secondary, tertiary, or quaternary amplification probes comprises a nucleotide sequence of at least 24 nucleotides in length. In some embodiments, each of the secondary, tertiary, or quaternary amplification probes comprises a nucleotide sequence of at least 25 nucleotides in length. In some embodiments, each of the secondary, tertiary, or quaternary amplification probes comprises a nucleotide sequence of at least 26 nucleotides in length.In some embodiments, the secondary, tertiary, or quaternary amplification probes each comprise a nucleotide sequence of at least 27 nucleotides in length. In some embodiments, the secondary, tertiary, or quaternary amplification probes each comprise a nucleotide sequence of less than 35, 40, 45, 50, 100, 150, 200, 250, or 300 nucleotides in length.

[0110] In some embodiments, the primary probe is amplified by contacting it with a secondary amplification probe. In some embodiments, the method includes contacting the secondary amplification probe with a tertiary amplification probe. In some embodiments, the method includes contacting the tertiary amplification probe with a quaternary amplification probe, wherein the quaternary amplification probe is identical to the secondary amplification probe.

[0111] In some embodiments, the method further includes amplifying one or more amplification probes by contacting one or more amplification probes from a previous contacting step with new plurality of amplification probes. In some embodiments, the new plurality of amplification probes are the same as the previous plurality of amplification probes.

[0112] Crosslinking

[0113] In some embodiments, the method includes crosslinking one or more amplification probes to one or more cellular components. In some embodiments, one or more amplification probes are crosslinked to one or more cellular components. In some embodiments, one or more amplification probes are crosslinked to proteins immediately adjacent to one or more cellular components. In some embodiments, one or more amplification probes are crosslinked to transcripts, RNA, DNA sites, chromosomes, DNA, proteins, lipids, glycans, cellular components, organelles, and any combination thereof immediately adjacent to one or more cellular components.

[0114] In some embodiments, one or more amplification probes are cross-linked to one or more cellular components via an optically dependent chemical moiety. In some embodiments, one or more amplification probes are cross-linked to proteins immediately adjacent to one or more cellular components via an optically dependent chemical moiety. In some embodiments, one or more amplification probes are cross-linked to transcripts, RNA, DNA sites, chromosomes, DNA, proteins, lipids, glycans, cellular components, organelles, and any combination thereof immediately adjacent to one or more cellular components via an optically dependent chemical moiety.

[0115] In some embodiments, the optically dependent chemical moiety is selected from diazididine, benzophenone, aryl azides, or any combination thereof. In some embodiments, the optically dependent chemical moiety is a photocrosslinker. In some embodiments, the amplification probe comprises one or more optically dependent chemical moieties.

[0116] In some embodiments, one or more amplification probes are cross-linked to one or more cellular components by exposure to light of a specific wavelength, specific intensity, specific duration, or any combination thereof. In some embodiments, one or more amplification probes are cross-linked to one or more cellular components via an ester. In some embodiments, the ester is an NHS-ester.

[0117] In some embodiments, one or more amplification probes are cross-linked to one or more cellular components using a cysteine-reactive reagent. In some embodiments, the reactive reagent is maleimide. In some embodiments, one or more amplification probes are cross-linked to one or more cellular components using a peptide conjugation reagent. In some embodiments, the peptide conjugation reagent is selected from carbodiimide, uronium salt, phosphonium salt, or any combination thereof.

[0118] In some implementations, one or more amplification probes are cross-linked to one or more cellular components via in-situ carbene generation.

[0119] In some implementations, one or more amplification probes are crosslinked to one or more cell components by contacting one or more cell components with one or more rounds of crosslinking agents.

[0120] In some embodiments, the crosslinking agent is selected from paraformaldehyde (PFA), polyethylene glycol bis(sulfosuccinimide) octanoate (BSPEG), or a combination thereof.

[0121] In some embodiments, one or more amplification probes are cross-linked to one or more cellular components by cross-linking cysteine ​​residues (via thiol-modified oligonucleotides). In some embodiments, one or more amplification probes are coupled with BM(PEG). n reaction.

[0122] In some embodiments, one or more amplification probes are cross-linked to one or more cellular components via amino oligonucleotides or thiol oligonucleotides. In some embodiments, one or more amplification probes are used via SM(PEG). n The lysine, cysteine, or a combination thereof are cross-linked to the cellular components.

[0123] In some embodiments, one or more amplification probes are crosslinked to one or more cellular components that are functionalized on a biomolecule with various reactive moieties (e.g., amino, hydroxyl, carboxylic acid, thiol, etc.), wherein the reactive moieties provide a reactive handle for the chemical deposition of the probes. In some embodiments, the cellular components are prepared to react with the reactive moieties on one or more amplification probes.

[0124] In some embodiments, one or more amplification probes are cross-linked to one or more cellular components via a cross-metathesis reaction. In some embodiments, the cross-metathesis reaction conjugates an olefin probe and an NHS-olefin handle.

[0125] In some embodiments, one or more amplification probes are crosslinked to one or more cellular components via N-halosuccinimide and olefinic amplification probes. In some embodiments, the crosslinking is performed using Heck coupling with a palladium catalyst.

[0126] In some embodiments, one or more amplification probes are cross-linked to one or more cellular components via a small molecule substrate coupled to a readout probe. In some embodiments, a riboswitch or a protein readout can be used to read out one or more amplification probes.

[0127] In some implementations, one or more amplification probes are crosslinked to one or more cellular components via a functionalized hydrogel containing functional groups that react with one or more modified amplification probes.

[0128] In some embodiments, one or more amplification probes are cross-linked to a primary probe. In some embodiments, one or more amplification probes are cross-linked to one or more primary probes. In some embodiments, one or more amplification probes are cross-linked at a location immediately adjacent to one or more cellular components. In some embodiments, one or more amplification probes are cross-linked at a location immediately adjacent to a primary probe that interacts with the target analyte.

[0129] In some embodiments, one or more amplification probes are crosslinked to cellular components via click chemistry. In some embodiments, the click chemistry may include, but is not limited to: strain-promoted azido-acetylene cycloaddition (SPAAC); tetrazine-trans-cyclooctene linkage (TCO-Tz; or reverse electron-demand Diels-Alder reaction); thiol-ene reaction; thiol-acetylene reaction; oxime linkage; hydrazone linkage; Diels-Alder reaction; and reverse electron-demand Diels-Alder reaction (IEDDA).

[0130] In some embodiments, the method includes contacting a cell sample with amine groups. In some embodiments, the amine groups in the cells are treated with Alkyne-PEG. N -NHS ester modification, where N is the number of monomer units. In some embodiments, the amine groups in the cells are modified with Azide-PEG. N -NHS ester modification.

[0131] In some implementations, thiols or mercapto groups are used as nucleophiles to perform cell functionalization to form the corresponding thioesters.

[0132] In some embodiments, the alkyne is present in the cell, for example, and can be crosslinked to the cell via a 3'-azide-modified probe by a copper (I)-catalyzed azido-alkyne cycloaddition (CuAAC).

[0133] In some embodiments, one or more amplification probes comprise 5' or 3' crosslinkable molecules. In some embodiments, the crosslinkable molecule is a photocrosslinkable molecule.

[0134] In some embodiments, the method includes crosslinking the probe to cellular components by treating the hydrazide with a carbonyl group to form a stable hydrazone.

[0135] Displacement and cutting

[0136] In some implementations, the method includes one or more replacement probes separating one or more amplification probes from one or more primary probes.

[0137] In some implementations, the method includes replacing one or more amplification probes from one or more primary probes by cutting the amplification probes.

[0138] In some embodiments, the method includes separating one or more amplification probes by washing, displacement, cutting, photocutting, chemical reduction, chemical degradation, enzymatic digestion, enzymatic reactions that modify the amplification probes, or any combination thereof.

[0139] In some embodiments, the method includes separating one or more amplification probes using one or more substitution probes. In some embodiments, the method includes competition between the one or more substitution probes and the interactions between one or more primary probes and one or more amplification probes.

[0140] In some implementations, the method includes separating one or more amplification probes by binding a high-affinity locked nucleic acid (LNA) or RNA probe.

[0141] In some embodiments, the method includes separating one or more amplification probes by binding to one or more substitution probes. In some embodiments, the method includes cross-linking one or more substitution probes to a target analyte. In some embodiments, the method includes one or more substitution probes containing binding sites, wherein the binding sites are inversely complementary to sequences on one or more amplification probes. In some embodiments, the method includes one or more substitution probes interacting with primary, secondary, tertiary, or quaternary binding sites of the amplification probes.

[0142] In some implementations, one or more replacement probes include one or more binding sites for binding to secondary, tertiary, and / or quaternary probes.

[0143] In some implementations, the method includes separating one or more amplification probes by light-based cleavage of one or more amplification probes.

[0144] In some embodiments, the method includes a washing step in which one or more cleaved amplification probes are separated from the analyte.

[0145] In some implementations, the method includes separating one or more amplification probes by photocutting of one or more amplification probes.

[0146] In some implementations, the method includes separating one or more amplification probes by cleaving disulfide bridges within secondary or tertiary probes that interact with the amplification probes.

[0147] In some implementations, the method includes isolating one or more amplification probes by cleaving a pH-dependent portion within a secondary or tertiary probe that interacts with one or more amplification probes.

[0148] In some embodiments, the method includes isolating one or more amplification probes by site-specific protease cleavage of one or more amplification probes by TEV protease, ribonuclease, TALEN, zinc finger protein, lipase or endoglucosidase.

[0149] In some embodiments, the method includes separating one or more amplification probes by electrochemical cleavage of one or more amplification probes.

[0150] In some embodiments, the method includes separating one or more amplification probes by cleavage at one or more recognition sites of the amplification probes, or by cleavage of the double-stranded probes with restriction endonucleases. In some embodiments, a uracil-specific excision reagent (USER) enzyme cleaves one or more amplification probes.

[0151] In some implementations, the method includes separating one or more amplification probes by modifying them with CRISPR Cas9.

[0152] In some embodiments, the method includes separating one or more amplification probes by cleaving one or more amplification probes with a target-specific endonuclease.

[0153] In some embodiments, the method includes separating one or more amplification probes by binding one or more displacement probes, the displacement probes being cross-linked to cellular components.

[0154] In some implementations, the method includes separating one or more amplification probes by partially or completely removing secondary or tertiary probes that interact with one or more amplification probes.

[0155] Read aloud

[0156] In some embodiments, the readout probe is selected from proteins, modified proteins, RNA, oligonucleotides, antibodies, antibody fragments, and combinations thereof. In some embodiments, the readout probe further comprises a detectable portion. In some embodiments, the detectable portion is a fluorophore.

[0157] In some embodiments, the readout probe comprises an oligonucleotide with a detectable portion.

[0158] In some embodiments, the readout probe comprises an oligonucleotide with a length of at least 5 nucleotides. In some embodiments, the readout probe comprises an oligonucleotide with a length of at least 6 nucleotides. In some embodiments, the readout probe comprises an oligonucleotide with a length of at least 7 nucleotides. In some embodiments, the readout probe comprises an oligonucleotide with a length of at least 8 nucleotides. In some embodiments, the readout probe comprises an oligonucleotide with a length of at least 9 nucleotides. In some embodiments, the readout probe comprises an oligonucleotide with a length of at least 10 nucleotides. In some embodiments, the readout probe comprises an oligonucleotide with a length of at least 11 nucleotides. In some embodiments, the readout probe comprises an oligonucleotide with a length of at least 12 nucleotides. In some embodiments, the readout probe comprises an oligonucleotide with a length of at least 13 nucleotides. In some embodiments, the readout probe comprises an oligonucleotide with a length of at least 14 nucleotides. In some embodiments, the readout probe comprises an oligonucleotide with a length of at least 15 nucleotides. In some embodiments, the readout probe comprises an oligonucleotide with a length of at least 16 nucleotides. In some embodiments, the readout probe comprises an oligonucleotide with a length of at least 17 nucleotides. In some embodiments, the readout probe comprises an oligonucleotide with a length of at least 18 nucleotides. In some embodiments, the readout probe comprises an oligonucleotide with a length of at least 19 nucleotides. In some embodiments, the readout probe comprises an oligonucleotide with a length of at least 20 nucleotides. In some embodiments, the readout probe comprises an oligonucleotide with a length of at least 21 nucleotides. In some embodiments, the readout probe comprises an oligonucleotide with a length of at least 22 nucleotides. In some embodiments, the readout probe comprises an oligonucleotide with a length of at least 23 nucleotides. In some embodiments, the readout probe comprises an oligonucleotide with a length of at least 24 nucleotides. In some embodiments, the readout probe comprises an oligonucleotide with a length of at least 25 nucleotides. In some embodiments, the readout probe comprises an oligonucleotide with a length of at least 26 nucleotides. In some embodiments, the readout probe comprises an oligonucleotide with a length of at least 27 nucleotides. In some embodiments, the readout probe comprises an oligonucleotide with a length of at least 28 nucleotides. In some embodiments, the readout probe comprises an oligonucleotide with a length of at least 29 nucleotides. In some embodiments, the readout probe comprises an oligonucleotide with a length of at least 30 nucleotides. In some embodiments, the readout probe of any of the foregoing embodiments comprises an oligonucleotide with a length of less than 35, 40, 45, 50, or 100 nucleotides.

[0159] In some embodiments, the readout probe comprises a sequence complementary to the primary probe. In some embodiments, the sequence complementarity comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0160] In some embodiments, the readout probe comprises oligonucleotides having the same sequence. In some embodiments, the readout probe comprises oligonucleotides having different sequences.

[0161] In some embodiments, the method includes generating a signal from in situ sequencing via ligation sequencing or synthesis sequencing. In some embodiments, a signal is generated by hybridizing sequencing primers with a primary probe, an amplification probe, or any combination thereof. In some embodiments, the sequencing reaction produces a fluorescent signal. In some embodiments, a signal is generated via in situ sequencing of an amplification probe.

[0162] In some embodiments, the method includes sequencing amplicones derived from the sequencing methods of the foregoing embodiments. In some embodiments, sequencing the amplicones maps the spatial locations of one or more cellular targets, one or more target analytes, or any combination thereof. In some embodiments, the method includes barcoding the spatial locations of target analytes, cellular targets, or any combination thereof by directly capturing the amplification products or by diffusing spatial barcodes into the sample to mark locations, generating barcoded amplicones that are extracted and sequenced to map the identity and location of molecules. In some embodiments, any of the foregoing embodiments is performed using a microscope slide-based technique.

[0163] In some embodiments, the method includes detecting a signal generated by the interaction of two amplicones. In some embodiments, an aptamer or bridging probe generates a signal when two amplification products are physically adjacent. In some embodiments, the aptamer or bridging probe is an oligonucleotide, a protein, or any combination thereof. In some embodiments, one or more aptamers generate a signal indicating the physical proximity of two or more analytes. In some embodiments, one or more aptamers generate a signal indicating the physical proximity of two or more primary probes. In some embodiments, two or more primary probes target the same target analyte. In some embodiments, amplification products from two or more primary probes generate a signal that acts as a coincidence detector. In some embodiments, the coincidence detector indicates whether the interaction between the primary probes is specific or non-specific. In some embodiments, a non-specific interaction between only one primary probe does not generate a signal.

[0164] In some embodiments, the method includes two or more primary probes that interact with different target analytes. In some embodiments, the method includes amplifying products from the primary probes. In some embodiments, the amplified products from the primary probes generate an enhanced-specific signal that acts as an overlap detector, indicating the physical proximity of one or more target analytes, one or more cellular components, or any combination thereof. In some embodiments, a single amplified product from one or more primary probes generates a signal if the target analytes are physically close. In some embodiments, the proximity between combinations of target analytes (e.g., nucleic acids, proteins, glycans, or any combination thereof) generates a detectable signal. In some embodiments, the proximity distance is less than 1, 2, 4, 5, 6, 7, 8, 9, or 10 nanometers. In some embodiments, the proximity distance that generates the signal can be adjusted by the length of the aptamer or bridging probe. In some embodiments, the aptamer or bridging probe is a nucleic acid. In some embodiments, the aptamer or bridging probe is 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides in length. In some embodiments, two or more primary probes target different target analytes, wherein proximity of the primary probes produces new analytes for further detection, amplification, or any combination thereof.

[0165] Fluorescent clusters

[0166] In some embodiments, the readout probe comprises a fluorophore. In some embodiments, the fluorophore is any fluorophore that is deemed suitable by those skilled in the art.

[0167] In some embodiments, the fluorophore includes, but is not limited to, fluorescein, rhodamine, Alexa Fluor, DyLight fluor, ATTO dye, or any analogues or derivatives thereof. In some embodiments, the detectable portion includes, but is not limited to: fluorescein and fluorescein chemical derivatives; eosin; carboxyfluorescein; fluorescein isothiocyanate (FITC); fluorescein phosphorus amide (FAM); erythrosine; Bengal rose red; fluorescein secreted from the bacterium *Pseudomonas aeruginosa*; methylene blue; laser dyes; and rhodamine dyes (e.g., rhodamine, rhodamine 6G, rhodamine B, rhodamine 123, auramine O, sulforhodamine 101, sulforhodamine B, and Texas Red).

[0168] In some embodiments, the fluorophores include, but are not limited to: ATTO dyes; acridine dyes (e.g., acridine orange, acridine yellow); Alexa Fluor; 7-aminoactinomycin D; 8-anilino-1-naphthalenesulfonate; auramine-rhodamine staining agent; benzanthrone; 5,12-bis(phenylethynyl)tetraphenyl; 9,10-bis(phenylethynyl)anthracene; black varnish; Brainbow; calcein; carboxyfluorescein; carboxyfluorescein diacetate succinimide ester; carboxyfluorescein succinimide ester; 1-chloro-9,10-bis(phenylethynyl)anthracene; 2-chloro-9,10-bis(phenylethynyl)anthracene; 2-chloro-9,10-diphenylanthracene; coumarin; anthocyanin dyes (e.g., anthocyanins such as Cy3 and Cy5, DiOC6, SYBR Green I); DAPI; dark quencher; DyLight Fluor; Fluo-4; FluoProbes; Fluorescent ketone dyes (e.g., Calcein, Carboxyfluorescein, Carboxyfluorescein diacetate succinimide, Carboxyfluorescein succinimide, Eosin, Eosin B, Eosin Y, Erythrosine, Fluorescein, Fluorescein isothiocyanate, Fluorescein phosphorimide, Indigo Yellow, Mercurochrome); Fluoro-Jade staining agent; Fura-2; Fura-2-acetoxymethyl ester; Green fluorescent protein; Hoechst staining agent; Indigo Yellow; Indo-1; Lucifer Yellow; Fluorescein; Phytocyanin; Optical brighteners; Oxazine dyes (e.g., Tar Violet, Nile Blue, Nile Red); Perylene; Phenanthridine dyes (ethidium bromide and propidium iodide); Flame red dyes; Phycobilins; Phycoerythrin; Phycoerythrin; Hydroxypyrene sulfonic acid; Rhodamine; Rhodamine 123; Rhodamine 6G; RiboGreen; RoGFP; Red fluorene; SYBR Green I; (E)-stilbene; (Z)-stilbene; sulforhodamine 101; sulforhodamine B; Synapto-pHluorin; tetraphenylbutadiene; tetrasodium tris(tetrasodium copper disulfonic acid)ruthenium(II); Texas Red; TSQ; umbelliferone; or yellow fluorescent protein.

[0169] In some embodiments, the fluorophores include, but are not limited to, the Alexa Fluor family of fluorescent dyes (Molecular Probes, Oregon). Alexa Fluor dyes are widely used as cell and tissue markers in fluorescence microscopy and cell biology. The excitation and emission spectra of Alexa Fluor cover the visible spectrum and extend into the infrared region. Members of this family are approximated according to their maximum excitation value (in nm). Some Alexa Fluor dyes are synthesized by sulfonating coumarin, rhodamine, xanthracenes (such as fluorescein), and anthocyanin dyes. In some embodiments, sulfonation gives the Alexa Fluor dye a negative charge and hydrophilicity. In some embodiments, Alexa Fluor dyes are more stable, brighter, and less pH sensitive than common dyes with comparable excitation and emission (e.g., fluorescein, rhodamine) and, to some extent, newer anthocyanin series. Exemplary Alexa Fluor dyes include, but are not limited to, Alexa-350, Alexa-405, Alexa-430, Alexa-488, Alexa-500, Alexa-514, Alexa-532, Alexa-546, Alexa-555, Alexa-568, Alexa-594, Alexa-610, Alexa-633, Alexa-647, Alexa-660, Alexa-680, Alexa-700, or Alexa-750.

[0170] In some embodiments, the fluorophore includes one or more of the DyLight Fluor family of fluorescent dyes (Dyomics and Thermo Fisher Scientific). Exemplary DyLight Fluor family dyes include, but are not limited to, DyLight-350, DyLight-405, DyLight-488, DyLight-549, DyLight-594, DyLight-633, DyLight-649, DyLight-680, DyLight-750, or DyLight-800.

[0171] In some embodiments, the fluorophore comprises nanomaterials. In some embodiments, the fluorophore is a nanoparticle. In some embodiments, the fluorophore is or comprises a quantum dot. In some embodiments, the fluorophore is a quantum dot. In some embodiments, the fluorophore comprises a quantum dot. In some embodiments, the fluorophore is or comprises gold nanoparticles. In some embodiments, the fluorophore is a gold nanoparticle. In some embodiments, the fluorophore comprises gold nanoparticles.

[0172] Imaging of the sample

[0173] In some embodiments, the method includes imaging the readout probe. In some embodiments, the method includes imaging the barcode. As will be understood by those skilled in the art, the imaging step may use different techniques.

[0174] In some embodiments, the imaging methods include, but are not limited to: epifluorescence microscopy, confocal microscopy, different types of super-resolution microscopy (PALM / STORM, SSIM / GSD / STED) and light sheet microscopy (SPIM, etc.).

[0175] In some embodiments, the imaging method includes exemplary super-resolution techniques, including but not limited to I... 5 M and 4Pi microscopy, stimulated emission loss microscopy (STEDM), ground state loss microscopy (GSDM), spatial structure illumination microscopy (SSIM), photoactivated localization microscopy (PALM), reversible saturable optical linear fluorescence transition (RESOLFT), total internal reflection fluorescence microscopy (TIRFM), fluorescence-PALM (FPALM), stochastic optical reconstruction microscopy (STORM), fluorescence imaging with one nanometer precision (FIONA) and combinations thereof. For example: Chi, 2009, “Super-resolution microscopy: breaking the limits,” Nature Methods 6(1): 15-18; Blow 2008, “New ways to see a smaller world,” Nature 456:825-828; Hell et al., 2007, “Far-Field Optical Nanoscopy,” Science 316: 1153; R. Heintzmann and G. Ficz, 2006, “Breaking the resolutionlimit in light microscopy,” Briefings in Functional Genomics and Proteomics 5(4):289-301; Garini et al., 2005, “From micro to nano: recent advances in high-resolution microscopy,” Current Opinion in Biotechnology 16:3-12; and Bewersdorf et al., 2006, “Comparison of I 5M and 4Pi-microscopy,” 222(2): 105-117; and Wells, 2004, “Man the Nanoscopes,” JCB 164(3):337-340.

[0176] In some implementations, an electron microscope (EM) is used for imaging.

[0177] In some implementations, the imaging step detects the target. In some implementations, the imaging step localizes the target. In some implementations, the imaging step provides three-dimensional spatial information of the target. In some implementations, the imaging step quantifies the target. By using multiple contact and imaging steps, the provided method is able to provide a large amount of spatial and / or quantitative information about a large number of targets with remarkably high throughput. For example, when using F different detectable markers, up to F0 can be obtained after N contact and imaging steps. N Spatial and / or quantitative information of each target.

[0178] Certain imaging techniques are known in the art. See, for example, International PCT Patent Application No. PCT / US2014 / 036258, filed April 30, 2014, entitled “Multi-labeling of molecules by sequential hybridization barcoding,” the entire contents of which are incorporated herein by reference for all purposes.

[0179] In some implementations, the method includes analyzing cell size and shape, markers, immunofluorescence measurements, or any combination thereof.

[0180] In some implementations, the signal is detected by imaging or sequencing. In some implementations, the sample is imaged after one or more amplification probes have been brought into contact with one or more readout probes.

[0181] Barcode the target

[0182] In some implementations, the method includes repeatedly contacting the amplification probe with one or more readout probes and an imaging step, each time using a new plurality of readout probes, such that the target analyte is described by a barcode and can be distinguished from another target analyte in the sample by the difference in its barcode.

[0183] In some embodiments, the target is selected from proteins, modified proteins, transcripts, RNA, DNA sites, exogenous proteins, exogenous nucleic acids, hormones, carbohydrates, small molecules, bioactive molecules, and combinations thereof. In some embodiments, the target includes subcellular features. For example, the nuclear lamina can be one set of barcodes, and the nucleolus can be targeted with another set of barcodes. This allows each sample to be analyzed using combinations of barcodes on different subcellular compartments. In some embodiments, the method includes barcoding the target, wherein the target is different.

[0184] In some embodiments, the method includes fluorescence detection. In some embodiments, the method includes fluorescence detection or other detection methods. In some embodiments, the method includes sequential hybridization to detect the target analyte.

[0185] In some embodiments, the probe is used in a method for barcoding one or more molecular targets. See, for example, International PCT Patent Application No. PCT / US2014 / 036258, filed April 30, 2014, entitled “Multi-labeling of molecules by sequential hybridization barcoding,” the entire contents of which are hereby incorporated herein by reference for all purposes.

[0186] In some embodiments, the probe is used in the exponentially radiometric anchored link amplification (LANTERN) method. See, for example, International Patent Application No. PCT / US2022 / 021826, filed March 24, 2022, entitled “Link amplification with exponentially radiometric anchoring,” the entire contents of which are incorporated herein by reference for all purposes.

[0187] In some implementations, the probe is used in the ClampFISH method. See, for example, ClampFISH uses click chemistry-based amplification to detect single nucleic acid molecules, Rouhanifard SH et al., Nature Biotechnology 37: 84-89 (2019), the entire contents of which are hereby incorporated by reference for all purposes.

[0188] In some embodiments, the method includes a readout probe selected from proteins, modified proteins, RNA, oligonucleotides, antibodies, antibody fragments, and combinations thereof.

[0189] In some embodiments, the method includes contacting each of one or more samples with a first plurality of readout probes, such that the probes interact with one or more targets. In some embodiments, the method includes imaging the samples after the first contact step to detect the interaction between the readout probes and their targets.

[0190] In some implementations, the method includes a contact step that differs from another contact step on a marking of at least one target.

[0191] In some implementations, the method includes a contact step, wherein each of the first plurality of probes with a detectable marker is marked with a detectable portion.

[0192] In some implementations, the method includes a contact step, wherein the probe of each detectable marker contains a detectable portion, and at least one contact step differs from another in that each target has a different detectable portion.

[0193] In some implementations, the method includes a contact step in which at least two different readout probes interact with a first target, and in which at least two different readout probes interact with a second target.

[0194] In some implementations, the readout probe contains a marker selected from two, three, or four different markers.

[0195] In some embodiments, the target barcode in the sample contains the amplified signal. In some embodiments, the target barcode in the sample contains the signal amplified by rolling circle, padlock, branched DNA, ClampFISH, LANTERN, or any combination thereof.

[0196] In some implementations, the method includes using readout probes, wherein each probe for a detectable tag contains the same detectable portion and the same sequence.

[0197] In some implementations, the method includes readout probes, each of which interacts with its target via one or more intermediate probes, each of which hybridizes with the target.

[0198] In some implementations, the method includes repeating the contact and imaging steps, each time using a new plurality of readout probes, such that a target in the sample is described by a barcode and can be distinguished from another target in the sample by the difference in its barcode.

[0199] In some implementations, the method includes an error correction wheel. See, for example, International Patent Application No. PCT / US2017 / 044994, filed August 0, 2017, entitled "Sequential Detection of Molecular Targets Based on Pseudo-Color Barcodes with Embedded Error Correction Mechanism," the entire contents of which are incorporated herein by reference for all purposes.

[0200] In some implementations, the method includes an error-correction wheel performed by selecting block codes such as Hamming codes, Reed-Solomon codes, Gray codes, or any combination thereof.

[0201] In some embodiments, the method of any of the foregoing embodiments further includes an error correction step. In some embodiments, the error correction step includes performing additional contact and imaging rounds before, between, or after steps (i)-(v).

[0202] Remove probe

[0203] In some embodiments, the method includes a step of removing readout probes after one or more imaging steps. In some embodiments, the step of removing readout probes includes contacting a plurality of readout probes with an enzyme that digests the readout probes. In some embodiments, the removal step includes contacting the plurality of readout probes with DNase, contacting the plurality of readout probes with RNase, photobleaching, strand displacement, formamide washing, thermal denaturation, or a combination thereof. In some embodiments, the removal step includes using photobleaching to remove the readout probes.

[0204] In some embodiments, the method includes removing the readout probe by using a stripping agent, a washing buffer, photobleaching, chemical bleaching, or any combination thereof.

[0205] In some embodiments, the amplification probe is separated by rigorous washing conditions of the sample. In some embodiments, the rigorous washing conditions include 30%, 40%, 50%, 55%, 60%, or 70% formamide in a buffer or aqueous solution.

[0206] In some embodiments, the method includes clarifying the sample. In some embodiments, the sample is clarified using CLARITY.

[0207] Certain techniques for removing probes are known in the art. See, for example, International PCT Patent Application No. PCT / US2014 / 036258, filed April 30, 2014, entitled “Multi-labeling of molecules by sequential hybridization barcoding,” the entire contents of which are hereby incorporated by reference for all purposes.

[0208] washing

[0209] In some embodiments, the methods of any of the foregoing embodiments optionally include washing the sample after each step. In some embodiments, the sample is washed with a buffer to remove nonspecific hybridization reactions. In some embodiments, formamide is used in the washing step. In some embodiments, the washing buffer is stringent. In some embodiments, the washing buffer contains 10% formamide, 2xSSC, and 0.1% Triton X-100.

[0210] After a detailed description of the embodiments, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope defined by the appended claims. Furthermore, it should be understood that all examples in this disclosure are provided as non-limiting examples.

[0211] The following non-limiting methods and examples are provided to further illustrate the implementations disclosed herein. Those skilled in the art will understand that the techniques disclosed in the following methods and examples represent methods that have been found to work well in practice and can therefore be considered examples constituting their mode of practice. However, those skilled in the art will understand upon reading this disclosure that many changes can be made to the specific implementations disclosed and similar or analogous results can still be obtained without departing from the spirit and scope of the implementation.

[0212] Example

[0213] Those skilled in the art will understand that the accompanying drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of this teaching in any way.

[0214] Example 1

[0215] The implementation is, for example Figure 1 As shown, an exemplary design for a linear amplification process is described.

[0216] Figure 1 Figure A illustrates an exemplary implementation of the binding of a primary probe to a target molecule. In this embodiment, the ssDNA primary probe binds to the RNA target through a site defined by the reverse complementary region of the primary probe.

[0217] Figure 1 Figure B illustrates an exemplary implementation where the amplification probe binds to a specific region of the primary probe. In this embodiment, the ssDNA amplification probe binds to a specific region of the primary ssDNA probe at a site where the two probes have reverse complementary sequences. The amplification probe is functionalized with reactive groups.

[0218] Figure 1 Figure C illustrates an exemplary embodiment in which an amplification probe binds to a sample via a reactive group. In this embodiment, the amplification probe group is modified with a photoreactive group that can be crosslinked with reactive groups in the sample by ultraviolet light excitation. After binding, the amplification probe is cleaved and partially washed away, or displaced from the primary probe, thereby releasing the initial primary probe binding site for another amplification probe.

[0219] Figure 1 Figure D illustrates an exemplary implementation where a subsequent amplification probe binds to a primary probe. In this embodiment, the amplification probe binds to the same released site where the previous amplification probe bound.

[0220] Figure 1 Figure E illustrates an exemplary implementation of multiple rounds of amplification probe binding to a primary probe, binding to the sample, and dissociating from the primary probe to allow the next amplification probe to bind to the same primary probe site. With each round, the number of amplification probes near the target molecule can increase by one. This process is repeated until a sufficient number of amplification probes for detecting the target molecule have accumulated in the sample.

[0221] Figure 1 Figure F illustrates an exemplary implementation where a readout probe binds to an amplification probe bound to a sample to detect a target molecule. In this embodiment, the readout probe is an ssDNA probe that binds to the amplification probe at a specific site where the two probes are inversely complementary to each other.

[0222] Example 2

[0223] The implementation is, for example Figure 2 The diagram illustrates an exemplary design where the probe is not bound to the sample. The method described herein cannot amplify the signal if the amplification probe is not repeatedly bound to the sample.

[0224] Figure 2 Figure A illustrates an exemplary implementation of primary probe binding to a target molecule. In this embodiment, the ssDNA primary probe binds to the RNA target at a site defined by the reverse complementary region of the primary probe. This is in contrast to... Figure 1 A describes the same implementation setup.

[0225] Figure 2 Figure B illustrates an exemplary implementation of the binding of the amplification probe to the binding region of the primary probe. In this embodiment, the ssDNA amplification probe binds to a specific region of the ssDNA primary probe at a site where the two probes have reverse complementary sequences. The amplification probe is functionalized with reactive groups. This is in contrast to... Figure 1 B describes the same implementation setup.

[0226] Figure 2 Figure C illustrates an exemplary implementation where the amplification probe is not bound to the sample. Because the amplification probe is not attached to the sample, the exemplary cleavage shown here results in the removal of the amplification probe from the sample.

[0227] Figure 2 Figure D illustrates an exemplary implementation where the readout probe does not bind to the sample, as shown by the dashed line. No amplification probe binds to the sample, and there are no binding sites available for the readout probe, thus the target molecule cannot be detected.

[0228] Example 3

[0229] The implementation is, for example Figure 3The diagram illustrates an exemplary design for a segmented linear amplification process.

[0230] Figure 3 Figure A illustrates an exemplary implementation where multiple amplification probes bind to a sample. After binding, each amplification probe is either cleaved and partially washed away, or displaced from the primary probe, thereby releasing the initial primary probe binding site from which another amplification probe binds. Multiple rounds of adding amplification probes result in multiple amplification probe rounds existing near the primary probe and the molecular target of the probe. This process is repeated until a sufficient number of amplification probes for detecting the target molecule have accumulated in the sample.

[0231] Figure 3 Figure B illustrates an exemplary embodiment where a secondary amplification probe contacts a primary probe bound to a sample. In this embodiment, the secondary amplification probe has a region that is reverse-complementary to the initially bound amplification probe. The number of secondary amplification probes that can bind to the sample depends on the number of initial amplification probes present in the sample. In this exemplary embodiment, the number of secondary amplification probes that can bind to the initial amplification probe is 6.

[0232] Figure 3 Figure C illustrates an exemplary embodiment of binding secondary amplification probes to a sample. This can be induced in the same or different manner as binding the initial amplification probe. In this exemplary embodiment, six secondary amplification probes are bound to the sample.

[0233] Figure 3 Figure D illustrates an exemplary implementation where six more secondary amplification probes are added during a subsequent round of secondary amplification probe addition, sample binding, and dissociation from the initial amplification probe.

[0234] Figure 3 Figure E illustrates an exemplary implementation where six more secondary amplification probes are added during a subsequent round of secondary amplification probe addition, sample binding, and dissociation from the initial amplification probe. The example process can be repeated until a sufficient number of secondary amplification probes are available for molecular target detection.

[0235] Figure 3 Figure F illustrates an exemplary embodiment in which each secondary amplification probe can be bound to a readout probe, which can be used to detect molecular targets in a sample. In this exemplary embodiment, the readout probe can be a fluorophore-conjugated ssDNA oligonucleotide sequence.

[0236] Example 4

[0237] The implementation is, for example Figure 4 As shown, an exemplary design of a combined linear and exponential amplification process is described.

[0238] Figure 4Figure A illustrates an exemplary implementation of multiple amplification probes binding to a sample. After binding, each amplification probe is either cleaved and partially washed away, or displaced from the primary probe, allowing another amplification probe to bind to the primary probe. Multiple rounds of adding amplification probes result in multiple amplification probes present near the primary probe and the molecular target of the probe. This process is repeated until a sufficient number of amplification probes for detecting the target molecule have accumulated in the sample.

[0239] Figure 4 Figure B illustrates an exemplary embodiment of a secondary amplification probe contacting a primary amplification probe bound to a sample. In this embodiment, the secondary amplification probe has a region that is reverse-complementary to the initially bound amplification probe. The number of secondary amplification probes that can bind to the sample depends on the number of initial amplification probes present in the sample. In this exemplary embodiment, the number of secondary amplification probes that can bind to the initial amplification probe is 6. This embodiment only shows a single step with 6 amplification probes. As previously described, the processes of binding, cleaving, or replacing, and binding additional amplification probes, can be repeated until a sufficient number of amplification probes for detecting the target molecule have accumulated in the sample. For exponential amplification in subsequent steps of this embodiment, each amplification probe bound here has two binding sites for amplification probes in the next round of amplification.

[0240] Figure 4 Figure C illustrates an exemplary implementation of binding exponential amplification probes to a sample. In this exemplary implementation, 12 tertiary amplification probes are bound to the 6 amplification probes bound to the sample in the previous step. In this embodiment, no probe displacement or cleavage occurs. Each amplification probe is bound to the sample and has two sites available for subsequent amplification probe binding.

[0241] Figure 4 Figure D illustrates an exemplary implementation of binding an exponential amplification probe to a sample.

[0242] Example 5

[0243] The implementation is, for example Figure 5 As shown, an exemplary design is described that follows an exponential amplification process based on the amplification factor according to the Pell sequence.

[0244] Figure 5Figure A illustrates an exemplary implementation of the binding of a primary probe to a target molecule and the binding of an amplification probe to a primary probe. In this embodiment, the ssDNA primary probe binds to the RNA target via a site defined by the inverse complementary region of the primary probe. The primary probe also has a binding feature R+ for the amplification probe, which binds to the primary probe via the inverse complementary feature R-. The dashed lines at the ends of the primary probes indicate that each primary probe typically contains multiple unique amplification probe binding sites, and are omitted for clarity in this example diagram. The first amplification probe has a feature that allows binding to the sample (circle), a cleavable feature (square), and two binding sites (A+) for the second amplification probe. In summary, in the method step labeled here as Round 1, there are a total of two binding features A+ in the sample.

[0245] Figure 5 Figure B illustrates an exemplary implementation where two secondary amplification probes bind at feature A- to a cross-linked and cleaved first amplification probe at feature A+. After cross-linking and cleaving, only the cross-linkable portion of the first amplification probe (including the binding site for the second amplification probe) remains in the sample. Note that the position of the cross-linked probe has been shifted in the illustration for clarity. Elements present prior to this round are shown in light gray for clarity. The secondary amplification probe has two binding features R+, which are designed to be identical to the binding features R+ on the primary probe.

[0246] Figure 5 Figure C illustrates an exemplary implementation of the next round, in which the same amplification probe as in the initial round is applied to the sample. This amplification probe binds to the secondary amplification probe at feature R+ via its binding feature R-. Similarly, the first amplification probe has two binding features A+. The number of newly added binding features A+ in this round of amplification is 10. The total number of available binding features A+ in the sample is now 12.

[0247] Figure 5 Figure D illustrates an exemplary implementation of another round of amplification using a secondary amplification probe, such as... Figure 5 B describes binding to the A+ site.

[0248] Figure 5 Figure E illustrates an exemplary implementation of the next round of amplification, in which the first amplification probe is added to the sample again. Combined with similar... Figure 5 As described in C. The number of newly added binding features A+ in this round of amplification is 58. The total number of available binding features A+ in the sample is now 70. This process can be repeated with additional rounds until a sufficient number of features A+ are bound to the sample. In this embodiment, features A+ can be bound by a fluorophore-conjugated readout probe. Alternatively, the readout probe can also be bound to any other amplification probe element shown in this embodiment.

[0249] Example 6

[0250] The implementation is, for example Figure 6 As shown, an exemplary design of the exponential amplification process is described.

[0251] Figure 6 Figure A illustrates an exemplary implementation of the binding of a primary probe to a target molecule and the binding of an amplification probe to the primary probe. In this embodiment, the ssDNA primary probe binds to the RNA target at a site defined by the reverse complementary region of the primary probe. The primary probe also has a binding feature R+ for the amplification probe, which can bind to the primary probe via the reverse complementary feature R-. The dashed lines at the ends of the primary probes indicate that each primary probe typically contains multiple unique amplification probe binding sites, and are omitted for clarity in this example diagram. The first amplification probe has a feature that allows binding to the sample (circle) and two binding sites (A+) for the second amplification probe. In summary, in the method step labeled here as Round 1, there are a total of two binding features A+ in the sample.

[0252] Figure 6 Figure B illustrates an exemplary embodiment in which two secondary amplification probes bind at feature A- to a cross-linked first amplification probe at feature A+. The secondary amplification probe has two binding features R+, which can be identical to the binding feature R+ of the primary probe, and is used for amplification. In this embodiment, no cleavage or replacement of the amplification probe occurs after cross-linking the secondary amplification probe. In summary, in the example method step labeled here as round 2, a total of four binding features R+ are added to the sample.

[0253] Figure 6 Figure C illustrates an exemplary implementation of four amplification probes, which can be the same as the amplification probes used in round 1, binding at feature R- to the cross-linked first amplification probe at feature R+. The amplification probes have two binding features A+. In this embodiment, no cleavage or displacement of the amplification probes occurs after cross-linking the secondary amplification probes. In summary, in the example method step labeled round 3 here, a total of 8 binding features A+ are added to the sample. This process is repeated until a sufficient number of amplification probes for detecting the target molecule have accumulated in the sample.

[0254] Example 7

[0255] The implementation is, for example Figure 7 As shown, an exemplary design of an amplification probe that can be bound to a sample is described.

[0256] Figure 7A diagram illustrates an exemplary implementation of a primary probe bound to an amplification probe. Details of an exemplary design for the amplification probe are given. Only one amplification probe is shown here; multiple identical or different amplification probes may bind to the primary probe. The amplification probe consists of a primary probe binding site (R-), which is the reverse complement of an amplification probe binding site (R+) on the primary probe. A feature (rectangular) that allows cleavage or probe displacement is part of the amplification probe and allows the release of the primary probe site after crosslinking to complete an amplification cycle. The cleavage site may also be located within the primary probe binding site R- or in other features of the amplification probe adjacent to the primary probe binding site R-. As a displacement site, R- may be a stalk sequence for the displacement strand, which is reverse complementary to the amplification probe. Other features of the amplification probe adjacent to the primary probe binding site R- may be additional stalk features for efficient displacement strand binding. The amplification probe further has an example binding site A+, which is bound by other amplification probes or readout probes that allow detection of signals, for example, by conjugating a fluorescent dye to the readout probe.

[0257] Figure 7 B illustrates a similar example. Figure 7 A is an exemplary implementation of the amplification probe, but it shows two binding sites A+ instead of one, for use in designs involving, for example, stronger linear amplification or nonlinear amplification schemes.

[0258] Example 8

[0259] The implementation is, for example Figure 8 As shown, an example design of a scheme using photocrosslinking and displacement from a primary probe to replace the probe is described.

[0260] Figure 8 Figure A illustrates an exemplary implementation of primary probe binding to a target molecule and amplification probe binding to a primary probe. In this embodiment, the primary probe has four binding sites R1+, R2+, R3+, and R4+, which can be bound by a unique amplification probe through their respective sites R1-, R2-, R3-, and R4-. The dashed rectangles indicate the following diagram. Figure 8 The primary probe portion of BD. The white circles at the ends of the amplification probes illustrate features that can bind (e.g., via UV crosslinking) to the sample.

[0261] Figure 8 Figure B illustrates an exemplary implementation showing a more detailed view of the binding of the amplification probe to the primary probe at the R3+ / R3- site. In addition to the binding site R3-, the example amplification probe has a stalk sequence binding site T+ and an amplification probe binding site A3+. The crossed circles in this example indicate that the amplification probe binds to the sample via a connectable feature.

[0262] Figure 8Figure C illustrates an exemplary implementation of the binding of a displacement probe and an amplification probe. In this embodiment, binding is assisted by the stalk feature T+ on the amplification probe, which makes the binding of the displacement probe R3+T- more favorable than the binding of the amplification probe at the binding site R- and the binding of the primary probe at R3+.

[0263] Figure 8 Figure D illustrates an exemplary implementation where the replacement probe remains attached to the amplification probe after the unbound replacement probe has been washed away or otherwise removed from the sample. In this step, the previously bound amplification probe cannot rebind to the R3+ / R3- site of the primary probe because the bound replacement probe blocks the binding site. Therefore, the R3+ site of the primary probe remains available for other amplification probes in subsequent binding rounds.

[0264] Figure 8 E illustrates an exemplary implementation where another amplification probe binds to the feature R3+ of the primary probe and binds to the sample via a connectable feature (crossed circle).

[0265] Example 9

[0266] The implementation is, for example Figure 9 As shown, an example design of a scheme using photocrosslinking and displacement from a primary probe to replace the probe is described.

[0267] Figure 9 Figure A illustrates an exemplary implementation of primary probe binding to a target molecule and amplification probe binding to a primary probe. In this embodiment, the primary probe has four binding sites R1+, R2+, R3+, and R4+, which can be bound by a unique amplification probe through their respective sites R1-, R2-, R3-, and R4-. The dashed rectangles indicate the following diagram. Figure 9 The primary probe portion of BD. The white circles at the ends of the amplification probes illustrate features that can bind (e.g., via UV crosslinking) to the sample.

[0268] Figure 9 Figure B illustrates an exemplary implementation showing a more detailed view of the binding of the amplification probe to the primary probe at the R3+ / R3- site. In addition to the binding site R3-, the example amplification probe has two amplification probe binding sites A3+. The crossed circles in this example indicate that the amplification probe binds to the sample via a connectable feature.

[0269] Figure 9Figure C illustrates an exemplary implementation of the binding of a substitution probe and an amplification probe. In this embodiment, binding is assisted by a stalk feature A3(1-n)-, which is the first n nucleotides of the A3- sequence that can bind to the A3+ sequence on the amplification probe. The length is, for example, 10 nt, so the total length of the substitution probe in this example is 25 nt. The stalk sequence makes the binding of the substitution probe R3+A3(1-n)- more favorable than the binding of the amplification probe at the binding site R- and the binding of the primary probe at R3+. Furthermore, the R3+A3(1-n)- substitution allows amplification probes with two A3+ feature sites to be flexibly used in exponential amplification rounds without substitution.

[0270] Figure 9 Figure D illustrates an exemplary implementation where the replacement probe remains attached to the amplification probe after the unbound replacement probe has been washed away or otherwise removed from the sample. In this step, the previously bound amplification probe cannot rebind to the R3+ / R3- site of the primary probe because the bound replacement probe blocks the binding site. Therefore, the R3+ site of the primary probe remains available for other amplification probes in subsequent binding rounds.

[0271] Figure 9 E illustrates an exemplary implementation where another amplification probe binds to the feature R3+ of the primary probe and binds to the sample via a connectable feature (crossed circle).

[0272] Example 10

[0273] The implementation is, for example Figure 10 As shown, an example design is provided for a scheme that uses photocrosslinking and amplification probe cleavage to dissociate the probe from the primary probe to replace the probe.

[0274] Figure 10 Figure A illustrates exemplary implementations of primary probe binding to target molecules and amplification probe binding to primary probes, similar to... Figure 9 A. In this embodiment, the primary probe has four binding sites R1+, R2+, R3+, and R4+, which can be bound by a unique amplification probe through their respective sites R1-, R2-, R3-, and R4-. The white circles at the ends of the amplification probes illustrate features that can bind (e.g., via UV crosslinking) to the sample.

[0275] Figure 10 Figure B illustrates an exemplary implementation showing a more detailed view of the binding of the amplification probe to the primary probe at the R3+ / R3- site. The crossed circles in this example indicate that the amplification probe binds to the sample via a connectable feature. The white rectangles illustrate examples of the cleavable features of the amplification probe.

[0276] Figure 10Figure C illustrates an exemplary embodiment of an amplification probe cleaved at a cleavable feature. The portion of the amplification probe containing the binding feature A+ remains near the primary probe because it is connected to the sample via a connectable feature indicated by a cross-shaped circle.

[0277] Figure 10 Figure D illustrates an exemplary implementation of the remaining portion of a cleaved amplification probe, which still binds to the example binding site R3+ via the binding feature R3-. This portion of the cleaved amplification probe can be removed, for example, by washing with conditions suitable for a short remaining fragment.

[0278] Figure 10 Figure E illustrates an exemplary implementation where another amplification probe binds to the primary probe's feature R3+ and binds to the sample via a connectable feature (circle), which... Figure 10 The steps described in B-10D become available.

[0279] Example 11

[0280] The implementation is, for example Figure 11 The image shown illustrates an example of the linear amplification process of fluorescence signal intensity of a single gene in a cell culture sample using diaziridine-modified oligonucleotides.

[0281] Approximately 100,000 NIH3T3 cells were seeded onto 24x60 mm #1.5 glass coverslips. Cells were fixed with 1x PBS in 4% PFA for 10 minutes at room temperature. After washing with 1x PBS, the samples were stored in 70% ethanol and incubated overnight at -20°C. The samples were dried with nitrogen and a custom flow cell was attached to the coverslips. After rinsing with 1x PBS, 7.5 mM BS-PEG5 in 1x PBS was added and incubated at room temperature for 30 minutes. The samples were incubated twice at room temperature for 30 minutes each time with 100 mM N-(propionyloxy)succinimide in 1x PBS. The samples were washed three times with 40% wash buffer (40% formamide, 2x SSC, 0.1% Triton-X100). The primary probe was hybridized to the sample at 37°C for 12 hours using 40% hybridization buffer (40% formamide, 0.1 mg / mL yeast tRNA, 2X SSC, 10% 500 kDa dextran sulfate) containing 5 nM / oligonucleotide. After hybridization, the sample was washed three times with 40% wash buffer and incubated once at 37°C. The sample was then washed with 2X SSC. Next, the 100 nM / oligonucleotide amplification probe was hybridized to the sample at room temperature for 30 minutes in 10% hybridization buffer (10% formamide, 2X SSC, 10% 6-10 kDa dextran sulfate, 0.1% Triton-X100). The sample was washed with 10% wash buffer (wash buffer: 10% formamide, 2X SSC, 0.1% Triton X-100) and 1x PBS, and then irradiated with a 365 nm UV lamp for 45 minutes. Next, the sample was washed with 2x SSC and incubated with 1 μM replacement strand in 15% hybridization buffer at room temperature for 15 minutes. After replacement, the sample was washed with 20% wash buffer (20% formamide, 2X SSC, 0.1% Triton X-100). The process of hybridization, cross-linking, and replacement of amplification probes was repeated multiple times to obtain sufficient signal amplification. Samples ready for imaging were hybridized for 15 minutes at room temperature using 10% hybridization buffer containing 100 nM / oligonucleotide readout probe. The samples were stained with 3 μg / mLDAPI in 2X SSC for 1 minute and then washed twice with 2X SSC. The samples were imaged using anti-quenching buffer (100 mM Tris-HCl pH 8, 4x SSC, 2 mM Trolox, 20% (w / v) D-glucose, 1:100 glucose oxidase (200 U / mL), 1:1000 catalase) and a 63x, 1.4 NA Leica objective lens and a confocal microscope (Andor Dragonfly).

[0282] Figure 11Figure A illustrates an exemplary implementation of increasing fluorescence intensity signal during multiple rounds of linear amplification.

[0283] Figure 11 Figure B illustrates an exemplary implementation of an increase in fluorescence intensity signal after five rounds of continuous linear amplification.

[0284] Figure 11 Figure C illustrates an exemplary implementation of an increase in fluorescence intensity signal after eleven rounds of continuous linear amplification.

[0285] Example 12

[0286] The implementation is, for example Figure 12 The image shown illustrates a linear amplification process of fluorescence signal intensity of a single gene in a cell culture sample using benzophenone-modified oligonucleotides. Scale bar: 20 micrometers.

[0287] Approximately 100,000 NIH3T3 cells were seeded onto 24x60 mm #1.5 glass coverslips. Cells were fixed with 1x PBS in 4% PFA for 10 minutes at room temperature. After washing with 1x PBS, the samples were stored in 70% ethanol and incubated overnight at -20°C. The samples were dried with nitrogen and a custom flow cell was attached to the coverslips. After rinsing with 1x PBS, 7.5 mM BS-PEG5 in 1x PBS was added and incubated at room temperature for 30 minutes. The samples were incubated twice at room temperature for 30 minutes each time with 100 mM N-(propionyloxy)succinimide in 1x PBS. The samples were washed three times with 40% wash buffer (40% formamide, 2x SSC, 0.1% Triton-X100). The primary probe was hybridized to the sample at 37°C for 12 hours using 40% hybridization buffer (40% formamide, 0.1 mg / mL yeast tRNA, 2X SSC, 10% 500 kDa dextran sulfate) containing 5 nM / oligonucleotide. After hybridization, the sample was washed three times with 40% wash buffer and incubated once at 37°C. The sample was then washed with 2X SSC. Next, the 100 nM / oligonucleotide amplification probe was hybridized to the sample at room temperature for 30 minutes in 10% hybridization buffer (10% formamide, 2X SSC, 10% 6-10 kDa dextran sulfate, 0.1% Triton-X100). The sample was washed with 10% wash buffer (wash buffer: 10% formamide, 2X SSC, 0.1% Triton X-100) and 1x PBS, and then irradiated with a 365 nm UV lamp for 45 minutes. Next, the sample was washed with 2x SSC and incubated with 1 μM replacement strand in 15% hybridization buffer at room temperature for 15 minutes. After replacement, the sample was washed with 20% wash buffer (20% formamide, 2X SSC, 0.1% Triton X-100). The process of hybridization, cross-linking, and replacement of amplification probes was repeated multiple times to obtain sufficient signal amplification. Samples ready for imaging were hybridized for 15 minutes at room temperature using 10% hybridization buffer containing 100 nM / oligonucleotide readout probe. The samples were stained with 3 μg / mLDAPI in 2X SSC for 1 minute and then washed twice with 2X SSC. The samples were imaged using anti-quenching buffer (100 mM Tris-HCl pH 8, 4x SSC, 2 mM Trolox, 20% (w / v) D-glucose, 1:100 glucose oxidase (200 U / mL), 1:1000 catalase) and a 63x, 1.4 NA Leica objective lens and a confocal microscope (Andor Dragonfly).

[0288] Figure 12An exemplary embodiment illustrating the increase in fluorescence intensity signal during multiple rounds of linear amplification of benzophenone-functionalized oligonucleotides is shown.

[0289] Example 13

[0290] Figure 13 The illustration shows an exemplary implementation of fluorescence intensity distribution changes during multiple rounds of linear amplification of benzophenone-functionalized oligonucleotides.

[0291] Example 14

[0292] The implementation is, for example Figure 14 The image shown illustrates example images depicting the linear and exponential amplification processes of combined fluorescence signal intensities of single genes in cell culture samples using benzophenone-modified oligonucleotides.

[0293] Approximately 100,000 NIH3T3 cells were seeded onto 24 x 60 mm #1.5 glass coverslips. Cells were fixed for 10 minutes at room temperature with a solution of 3% glyoxal, 0.8% acetic acid, 150 mM NaCl, and 45 mM NaOH. After washing with 1x PBS, the samples were stored in 70% ethanol and incubated overnight at -20°C. The samples were dried with nitrogen, and a custom flow cell was attached to the coverslip. After rinsing with 1x PBS, the samples were incubated with 0.1% NaBH4 for 15 minutes, followed by incubation twice for 15 minutes each time at room temperature with 7.5 mM BS-PEG5 in 1x PBS. The samples were then incubated twice for 15 minutes each time with 100 mM N-(propionyloxy)succinimide in 1x PBS at room temperature. The samples were washed three times with 50% wash buffer (50% formamide, 2x SSC, 0.1% Triton-X100). The primary probe was hybridized to the sample at 37°C for 16 hours using 50% hybridization buffer (50% formamide, 0.1 mg / mL yeast tRNA, 2X SSC, 10% 500 kDa dextran sulfate) containing 5 nM / oligonucleotide. After hybridization, the sample was washed three times with 55% wash buffer and incubated once at 37°C. Subsequently, the sample was washed with 4X SSC solution. Then, the amplification probe modified with 50 nM / oligonucleotide benzophenone, containing a 15 nt extension strand (the inverse complementary sequence between the toe and readout site to partially double-strand the amplification probe), was hybridized to the sample at 37°C for 45 minutes. The sample was washed with 15% wash buffer (wash buffer: 15% formamide, 2X SSC, 0.1% Triton X-100) and 1x PBS, and then irradiated with a 365 nm UV lamp for 5 min. Next, the sample was washed with 55% wash buffer (55% formamide, 2x SSC, 0.1% Triton X-100), then with 4x SSC, and incubated with 1 uM replacement strand in 10% hybridization buffer at room temperature for 15 min. After replacement, the sample was washed with 25% wash buffer (25% formamide, 2X SSC, 0.1% Triton X-100). In this example experiment, the process of hybridization, cross-linking, and replacement of the amplification probe was repeated 8 times to obtain sufficient signal amplification in the linear amplification step. The linear amplification probe was targeted in the exponential amplification rounds. For exponential amplification rounds, amplification probes modified with 50-100 nM oligonucleotide benzophenone with a 15 nt extension chain were hybridized with the sample for 45 minutes in 10% hybridization buffer (10% formamide, 2X SSC, 10% 6-10 kDa dextran sulfate, 0.1% Triton-X100).The sample was washed with 15% wash buffer (wash buffer: 15% formamide, 2X SSC, 0.1% Triton X-100) and 1x PBS, and then irradiated with a 365 nm UV lamp for 5 min. The sample was then washed with 55% wash buffer (55% formamide, 2X SSC, 0.1% Triton X-100), and then hybridized with the benzophenone-modified amplification probe for the next round. This procedure was repeated 18 times in this example experiment, with various imaging steps performed during the process. Samples prepared for imaging were hybridized for 15 min at room temperature using 10% hybridization buffer containing 100 nM / oligonucleotide readout probe. The sample was stained with 3 μg / mL DAPI in 2X SSC for 1 min, and then washed twice with 4X SSC. The samples were imaged using an anti-quenching buffer (100 mM Tris-HCl pH 8, 4x SSC, 2 mM Trolox, 10% (w / v) D-glucose, 1:100 glucose oxidase (200 U / mL), 1:1000 catalase) and a 63x, 1.4 NA Leica objective lens and a confocal microscope (Andor Dragonfly). Figure 14 Illustration A illustrates an exemplary implementation of increased fluorescence intensity signal. The markers above the example image indicate the total number of amplification rounds. The contrast of the example image has been adjusted to illustrate the similarity of fluorescence signal characteristics (such as spot size and number of spots) across different amplification rounds. Each spot corresponds to the signal of a single nucleic acid molecule. Figure 14 Figure B illustrates an exemplary implementation of increased fluorescence intensity signal during multiple rounds of amplification of benzophenone-functionalized oligonucleotides. In this example image, the peak intensity of a single point is plotted for each imaging round, and the fold increase in fluorescence intensity is written above the corresponding data point.

[0294] Example 15

[0295] Figure 15 The diagram illustrates an additional design for crosslinking amplification. Figure 15Figure A illustrates a schematic of the sacrificial layer design used for amplification. Primary probes hybridize to RNA molecules, followed by secondary and tertiary amplification probe hybridization. In this scenario, secondary amplification probes do not have cross-linking groups, but tertiary amplification probes do. After cross-linking the tertiary probes, a rigorous stripping step (60% formamide wash) removes the secondary amplification probes. Other uncross-linked probes, such as the primary probe, are also removed. RNA may also be degraded without affecting subsequent results. Additional secondary and tertiary hybridization steps are performed, where secondary amplification probes hybridize to the tertiary amplification probes already cross-linked into the cells. Tertiary amplification probes can hybridize to secondary amplification probes. Another rigorous washing step removes the secondary amplification probes. This results in more tertiary amplification probes cross-linking near the target analyte. After n rounds of hybridization, cross-linking, and washing, the number of tertiary amplification probes near the target analyte increases. Figure 15 Diagram B illustrates the implementation. Figure 15 Signal quantification of a single amplification site in cells according to scheme A. An average 16.7-fold amplification was achieved after three rounds of secondary and tertiary hybridization, crosslinking, and washing. Images show three amplification probes in NIH 3T3 cells using Eef2 as the target mRNA (D). Amplification probe 1 was quantified. Each secondary amplification probe has two binding sites for the tertiary probe, and each tertiary amplification probe has two binding sites for the secondary amplification probe. Histograms show the intensity of a single site in the cell compared to unamplified single-molecule FISH (smFISH) imaging. In this implementation, the tertiary amplification probe was crosslinked using a click reaction. Figure 15 Figure C illustrates a bridging aptamer design that generates a signal only when two amplification spheres are physically close to each other. The bridging probes, highlighted in bold, show the bridging aptamers binding across the two amplification spheres. Bold lines indicate readout probe binding sites. Amplification is performed with or without a sacrificial amplification layer. Primary probes are designed to hybridize to adjacent regions on the target analyte (RNA, shown in the figure). Each primary probe contains an amplification probe binding site, such that two amplification spheres are generated from adjacent amplification probe pairs. Figure 15 Diagram D illustrates the use Figure 20Cell images amplified using the protocol shown in C. 24 pairs of primary probes were used to target Eef2 mRNA in mammalian cell culture (NIH 3T3). Each pair contains primary probes that hybridize to adjacent regions separated by one nucleotide on the mRNA. Each primary probe contains two amplification probe binding sites, for a total of four sites. Three sites were amplified. Amplification probes 1 (secondary and tertiary) and 2 (secondary and tertiary) were located on one primary probe, while amplification probe 3 (secondary and tertiary) was located on an adjacent primary probe. Amplification was performed using a sacrificial secondary layer, which was removed by washing, while the tertiary amplification probe was cross-linked into the cells via a click reaction. Numerous spots were co-localized between amplification probes 1–3, indicating accurate amplification and detection of individual mRNA molecules in the cells. Bridge aptamers across amplification spheres generated by amplification probes 2 and 3 detected most of the same spots co-localized in the three amplification probe channels. Furthermore, non-specific amplification spots in each amplification channel (three images on the left) were not observed in the bridge aptamer channel (image on the right). A magnified image shown in a white box is displayed in the lower right corner of each image. The arrows in the lower right corner illustration show regions where nonspecific points appear in a single amplification channel but not in a bridge channel. Nonspecific amplification occurs because amplification probes (secondary or tertiary probes) can adhere nonspecifically to cells rather than to the actual analyte target. These nonspecific events can be further amplified, producing signals equivalent to the real signal. However, because nonspecific events are random, they are unlikely to occur at the same location for two different amplification probe sequences, such as amplification probe 2 and amplification probe 3. Using a bridge aptamer, which requires two or more amplification spheres to be present in close proximity, acts as a concordance detector, rejecting nonspecific binding and detecting only target analytes where the two primary probes and amplification products are physically close together. When applied to different analytes present in close proximity, such as RNA-DNA, RNA-protein, DNA-protein, protein-protein, or other molecules, this approach enables the detection of molecular interactions using amplified and bridged readout probes.

[0296] Example 16

[0297] The implementation is, for example Figure 16 The image illustrates example images depicting the linear and exponential amplification process of combined fluorescence signal intensities of single genes in cell culture samples using benzophenone-modified oligonucleotides. Cells were prepared as described in Example 14. Figure 16 Illustration A illustrates an exemplary implementation of recording a small fraction of an image showing an increase in fluorescence intensity signal to visualize a single fluorescence intensity peak. Each point corresponds to the signal of a single nucleic acid molecule. The markers above the example images indicate the total number of amplification rounds. All example images are contrast-matched, with the two rightmost images using a 1 / 10th exposure time to illustrate the increase in signal intensity. Figure 16Figure B illustrates an exemplary implementation of increased fluorescence intensity signal during multiple rounds of amplification of benzophenone-functionalized oligonucleotides. In this example image, the peak intensity of a single point is plotted for each imaging round, and the fold increase in fluorescence intensity is written above the corresponding data point.

[0298] Examples 17-20 describe RNA signal stabilization and amplification via click chemistry.

[0299] Example 17

[0300] In this embodiment, in one implementation, cell samples are prepared and fixed by modifying the amine groups in the cells using, for example, alkyne-PEG4-NHS esters through the activation of ester groups. For cell functionalization, in addition to adding functional groups to the cells via esterification, it can also be achieved using thiols or mercapto nucleophiles to produce the corresponding thioesters. In another implementation, alkyne groups are linked to the cells, wherein 3'-azide-modified DNA oligonucleotides can be crosslinked into the cells via a copper (I)-catalyzed azid-alkyne cycloaddition (CuAAC) reaction, preserving spatial information and amplifying signals of unstable RNA, such as... Figure 17 As shown in Figure A. For probe crosslinking, in addition to the CuAAC reaction described above, in one embodiment, other established click chemistry methods can also be used to achieve crosslinking, including but not limited to: strain-promoted azido-alkyne cycloaddition (SPAAC); tetrazine-trans-cyclooctene linkage (TCO-Tz; or reverse electron-demand Diels-Alder reaction); thiol-ene reaction; thiol-alkyne reaction; oxime linkage; hydrazone linkage; Diels-Alder reaction; and reverse electron-demand Diels-Alder reaction (IEDDA). Furthermore, the probe can be crosslinked into cells by treating the carbonyl group with an acylhydrazine to form, for example, a stable hydrazone.

[0301] Three amplification schemes based on DNA probe click-crosslinking to cells were developed: split design, branching design, and padlock design. For all three schemes, we designed two sets of amplification probes, referred to as secondary and tertiary amplification probes. Tertiary amplification probes can only bind to secondary amplification probes, and vice versa. This design prevents the amplification probes from accumulating in the hybridization solution before reaching the amplification site and achieves highly controllable amplification folds.

[0302] Example 18

[0303] To eliminate noise, a design was made such as Figure 18The split probes are shown in Figure A. Binding of each tertiary amplification probe requires both secondary amplification probes to be in close proximity, and binding of each secondary amplification probe requires either a primary probe pair or both tertiary amplification probes to be in close proximity. This "double-lock" system significantly reduces noise caused by random amplification probe adhesion to cells and subsequent amplification, as the likelihood of amplification probe pairs adhering to the same cellular site is extremely low. The 3' ends of the DNA oligonucleotides (primary and amplification probes) are modified with azide-modified dATP using terminal deoxynucleotidyl transferase (TdT). In this example, cell samples are prepared and fixed by modifying the amine groups in the cells with alkyne-PEG4-NHS ester through the activation of the ester groups. After each round of amplification probe hybridization, the sample is washed with 20% formamide wash buffer (20% formamide, 0.1% Triton X-100, 4x SSC) to remove excess amplification probes before clicking onto cells.

[0304] Evaluation Figure 17 The CuAAC crosslinking efficiency is shown in Figure B. The secondary amplification probes crosslinked in the first round were stripped using 60% formamide wash buffer (60% formamide, 0.1% Triton X-100, 4x SSC). Uncrosslinked amplification probes were washed away at room temperature, as shown in Figure B. Figure 17 As shown in Figure C, the signal intensity before and after peeling was quantified. For the "clicked" sample, the signal intensity did not decrease significantly, while the "unclicked" sample showed almost undetectable signal, as shown in Figure C. Figure 17 As shown in C-17D.

[0305] like Figure 18 As shown in B, this amplification protocol can be used to preserve and amplify RNA signals. Using a standard confocal microscope (Andor Dragonfly) at 10% laser power for 500 ms, the signal can reach 5000 counts, while maintaining good co-localization with the smFISH signal.

[0306] Example 19

[0307] An efficient amplification protocol was developed, allowing for rapid branching of short amplification probes. The RNA binding site on the primary probe was designed to be 30 nt, and the amplification probe binding sites were designed to be 13 nt / 15 nt, located at both ends of the primary probe. After hybridization, excess probe was removed using 30% formamide wash buffer (30% formamide, 0.1% Triton X-100, 4x SSC). The primary probe was then anchored to the cell at the 3' end using CuAAC. Signal amplification was achieved using a branched amplification probe containing a 13 nt / 15 nt probe binding site at the 5' end and two 13 nt / 15 nt amplification sites at the 3' end. To minimize noise, a 10% formamide wash was applied after each round of amplification probe hybridization. In this example, cell samples were prepared and fixed by modifying the amine groups in the cells with alkyne-PEG4-NHS ester to activate the ester groups. The amplification probe was then crosslinked to the cells via CuAAC to establish a branched structure. Excess amplification probe was removed after crosslinking using 60% formamide wash buffer. For rapid amplification using short-branched amplification probes, each round of probe hybridization can be reduced from 1 hour to 30 minutes. The amplification protocol is as follows: Figure 19 As shown in Figure A.

[0308] Amplification efficiency was tested using 24 Eef2 primary probes. A branching protocol was used to process the samples before signal amplification. After 6 rounds of amplification, we achieved approximately 18-fold signal enhancement with 13 nt amplification probe binding and approximately 38-fold signal enhancement with 15 nt amplification probe binding compared to smFISH. Figure 19 D). The highly amplified sites co-localized very well in both 13 nt BS and 15 nt BS designs, such as Figure 19 As shown in BC. Therefore, we have demonstrated that the branching scheme can achieve rapid and high-precision amplification.

[0309] Example 20

[0310] In this protocol, all probes are designed with a padlock configuration to allow for exonuclease digestion and reduce noise. The primary probes are designed with 21 nt RNA binding sites at each end of the padlock. After hybridization, excess probes are washed away with 30% formamide wash buffer (30% formamide, 0.1% Triton X-100, 4x SSC) and digested with exonucleases. Subsequently, the primary probes are cross-linked into cells using CuAAC.

[0311] The signal was then amplified using an azide-modified padlock amplification probe, which has 15 nt probe binding sites at each end and two 30 nt amplification sites in the middle for exponential amplification. To reduce noise, the amplification probes were washed with 10% formamide and digested with exonuclease after each round of hybridization. These amplification probes were also cross-linked into cells via a CuAAC reaction to construct the structure. After cross-linking, excess and uncross-linked amplification probes were washed away with 60% formamide wash buffer. The amplification protocol is as follows: Figure 20 As shown in Figure A.

[0312] The co-amplification of different sites on the same primary probe was tested. A single probe targeting the Eef2 gene was designed with three amplification probe binding sites. After 14 rounds of amplification, the signals in different channels co-localized very consistently, such as... Figure 20 As shown in B. Furthermore, we quantized the point intensity, achieving approximately 8x signal enhancement compared to standard smFISH with 24 primary probes. Figure 20 C). The primary probe is expected to have an 80% binding efficiency, therefore the amplification fold of a single probe is estimated to be approximately 150-fold. The strong colocalization and significant signal amplification in the single-probe experiments enable the detection of short RNAs and large-scale barcoding experiments.

[0313] References

[0314] Further background information can be found in the following references, each of which is incorporated into this paper in its entirety by citation.

[0315] 1. Moses, L. & Pachter, L. Museum of spatial transcriptomics. NatMethods 19, 534–546 (2022).

[0316] 2. Rouhanifard, SH et al. ClampFISH detects individual nucleic acid molecules using click chemistry–based amplification. Nat Biotechnol 37,84–89 (2019).

[0317] 3. Dardani, I. et al. ClampFISH 2.0 enables rapid, scalable amplifiedRNA detection in situ. Nat Methods 19, 1403–1410 (2022).

[0318] 4. Wu, C. et al. RollFISH achieves robust quantification of single-molecule RNA biomarkers in paraffin-embedded tumor tissue samples. CommunBiol 1, 1–8 (2018).

[0319] 5. Wang, X. et al. Three-dimensional intact-tissue sequencing ofsingle-cell transcriptional states. Science 361, eaat5691 (2018).

[0320] 6. Zeng, H. et al. Integrative in situ mapping of single-celltranscriptional states and tissue histopathology in a mouse model ofAlzheimer’s disease. Nat Neurosci 26, 430–446 (2023).

[0321] 7. Gyllborg, D. et al. Hybridization-based in situ sequencing(HybISS) for spatially resolved transcriptomics in human and mouse braintissue. Nucleic Acids Research 48, e112 (2020).

[0322] 8. Lee, J. H. et al. Highly Multiplexed Subcellular RNA Sequencing inSitu. Science 343, 1360–1363 (2014).

[0323] 9. Shah, S., Lubeck, E., Zhou, W. & Cai, L. In Situ TranscriptionProfiling of Single Cells Reveals Spatial Organization of Cells in the MouseHippocampus. Neuron 92, 342–357 (2016).

[0324] 10. Choi, H. M. T., Beck, V. A. & Pierce, N. A. Next-Generation inSitu Hybridization Chain Reaction: Higher Gain, Lower Cost, GreaterDurability. ACS Nano 8, 4284–4294 (2014).

[0325] 11. Xia, C., Babcock, H. P., Moffitt, J. R. & Zhuang, X. Multiplexeddetection of RNA using MERFISH and branched DNA amplification. Sci Rep 9,7721 (2019).

[0326] 12. He, S. et al. High-plex Multiomic Analysis in FFPE atSubcellular Level by Spatial Molecular Imaging. (2022) doi: https: / / doi.org / 10.1101 / 2021.11.03.467020

Claims

1. A method comprising: (a) contacting one or more target analytes in a sample with a plurality of primary probes, wherein each probe of the plurality of primary probes interacts with at least one target analyte; (b) contacting each of the plurality of primary probes, each of which interacts with at least one analyte, with one or more amplification probes; (c) optionally, cross-linking one or more amplification probes to a cellular component; (d) optionally, separating each of the one or more amplification probes from its primary probe or from another amplification probe; (e) optionally, repeating steps (b), (c), and / or (d); (f) detecting one or more target analytes.

2. The method of claim 1, further comprising contacting one or more amplification probes with one or more readout probes.

3. The method of claim 1, wherein the signal is detected by imaging or sequencing.

4. The method of claim 1, wherein the method comprises at least one step of cross-linking one or more amplification probes to a cellular component.

5. The method of claim 4, wherein the cross-linking step is by click chemistry.

6. The method of claim 5, wherein the click chemistry is selected from the group consisting of strain-promoted azide-alkyne cycloaddition (SPAAC), tetrazine-trans-cyclooctene ligation (TCO-Tz), thiol-ene reaction, thiol-yne reaction, oxime ligation, hydrazone ligation, Diels-Alder reaction; and inverse electron demand Diels-Alder reaction (IEDDA).

7. The method of claim 1, wherein the method comprises at least one step of separating each of the one or more amplification probes from its primary probe or from another amplification probe.

8. The method of claim 1, wherein one or more target analytes are detected by a signal or absence of a signal.

9. The method of claim 1, further comprising contacting one or more amplification probes with another amplification probe.

10. The method of claim 1, wherein one or more amplification probes comprise secondary, tertiary, and quaternary amplification probes.

11. The method of claim 10, wherein the quaternary amplification probe is the same as the secondary amplification probe.

12. The method of claim 10, wherein the quaternary amplification probe interacts with the secondary amplification probe.

13. The method of claim 10, wherein the quaternary amplification probe comprises one or more binding sites for the secondary amplification probe, one or more binding sites for the tertiary amplification probe, or any combination thereof.

14. The method of claim 10, wherein the tertiary amplification probe comprises one or more binding sites for the secondary amplification probe, one or more binding sites for the quaternary amplification probe, or any combination thereof.

15. The method of claim 10, wherein the secondary amplification probe comprises one or more binding sites for the tertiary amplification probe, one or more binding sites for the quaternary amplification probe, one or more binding sites for the primary probe, or any combination thereof.

16. The method of claim 1, wherein the primary probe is amplified by: (a) contacting the primary probe with a secondary amplification probe, (b) contacting the secondary amplification probe with a tertiary amplification probe, and (c) contacting the tertiary amplification probe with a quaternary amplification probe, wherein the quaternary amplification probe is the same as the secondary amplification probe.

17. The method of claim 1, wherein the sample is imaged after contacting one or more amplification probes with one or more readout probes.

18. The method of claim 1, further comprising amplifying one or more amplification probes by contacting the one or more amplification probes from a previous contacting step with a new plurality of amplification probes.

19. The method of claim 18, wherein the new plurality of amplification probes is the same as the previous plurality of amplification probes.

20. The method of claim 1, further comprising: (a) repeating the step of contacting one or more amplification probes with one or more readout probes and the imaging step, each time using a new plurality of readout probes, such that the target analyte is described by a barcode and can be distinguished from another target analyte in the sample by the difference in its barcode; and (b) optionally, separating the one or more amplification probes from the primary probe or another amplification probe after imaging the sample, (c) optionally, separating the readout probes from the amplification probes.

21. The method of claim 1, wherein the target analyte is selected from the group consisting of a transcript, an RNA, a DNA site, a chromosome, a DNA, a protein, a peptide, a lipid, a glycan, a cellular component, a small molecule, a metabolite, a primary probe, an amplification probe, an organelle, and any combination thereof.

22. The method of claim 1, wherein the cellular component is the target analyte.

23. The method of claim 1, wherein the cellular component is different from the target analyte.

24. The method of claim 7, wherein the cellular component is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 nanometers from the target analyte.

25. The method of claim 1, wherein the primary probe comprises one or more analyte binding sites.

26. The method of claim 25, wherein the analyte binding sites on the primary probe are the same as one another.

27. The method of claim 25, wherein the analyte binding sites on the primary probe are different from one another.

28. The method of claim 1, wherein the primary probe comprises one or more amplification probe binding sites.

29. The method of claim 28, wherein the amplification probe binding sites are identical to each other.

30. The method of claim 28, wherein the amplification probe binding sites are different from each other.

31. The method of claim 1, wherein the amplification probe comprises one or more primary probe binding sites.

32. The method of claim 1, wherein the amplification probe comprises a primary probe binding site, a moiety that allows the amplification probe to dissociate from the primary probe, one or more identical amplification probe binding sites, one or more different amplification probe binding sites, one or more identical readout probe binding sites, one or more different readout probe binding sites, one or more different cross-linking sites, a secondary probe binding site, a tertiary probe binding site, or any combination thereof.

33. The method of claim 1, wherein one or more amplification probes are cross-linked to the cellular component by one or more photo-dependent chemical moieties.

34. The method of claim 33, wherein the photo-dependent chemical moiety is selected from the group consisting of diazirine, benzophenone, aryl azide, or any combination thereof.

35. The method of claim 33, wherein one or more amplification probes are cross-linked to one or more cellular components by exposure to light of a particular wavelength, a particular intensity, a particular duration, or any combination thereof.

36. The method of claim 1, wherein one or more amplification probes are cross-linked to the cellular component by an ester.

37. The method of claim 36, wherein the ester is an NHS-ester.

38. The method of claim 1, wherein one or more amplification probes are cross-linked to the cellular component by a cysteine reactive reagent.

39. The method of claim 38, wherein the reactive reagent is a maleimide.

40. The method of claim 1, wherein one or more amplification probes are cross-linked to the cellular component by a peptide coupling reagent.

41. The method of claim 40, wherein the peptide coupling reagent is selected from the group consisting of carbodiimide, a uronium salt, a phosphonium salt, or any combination thereof.

42. The method of claim 1, wherein one or more amplification probes are cross-linked to the cellular component by in situ carbene generation.

43. The method of claim 1, wherein one or more amplification probes are cross-linked to the cellular component by contacting the analyte with one or more rounds of a cross-linking reagent.

44. The method of claim 43, wherein the cross-linking reagent is selected from the group consisting of paraformaldehyde (PFA), polyethylene glycolated bis(sulfosuccinimidyl)suberate (BSPEG), or a combination thereof.

45. The method of claim 1, wherein one or more amplification probes are cross-linked to the cellular component by cross-linking cysteines with oligonucleotides having thiol modifications.

46. The method of claim 45, further comprising allowing the amplification probe to react with BM(PEG) n .

47. The method of claim 1, wherein one or more amplification probes are crosslinked to the cellular component via an amine-oligonucleotide or a thiol-oligonucleotide.

48. The method of claim 47, further comprising using SM(PEG) n reacting lysine, cysteine, or a combination thereof.

49. The method of claim 1, wherein one or more amplification probes are crosslinked to the cellular component, which is functionalized with various reactive moieties on biomolecules, wherein the reactive moieties provide reactive handles for chemical deposition of probes.

50. The method of claim 1, wherein the cellular component is prepared to react with reactive moieties on the one or more amplification probes.

51. The method of claim 1, wherein one or more amplification probes are crosslinked to the cellular component via a cross-metathesis reaction.

52. The method of claim 51, wherein the cross-metathesis reaction conjugates an olefin probe and an NHS-olefin handle.

53. The method of claim 1, wherein one or more amplification probes are crosslinked to the cellular component via a Heck coupling using a palladium catalyst, via a reaction of N-halosuccinimide and an olefin.

54. The method of claim 53, wherein one or more amplification probes are crosslinked to the cellular component via a small molecule substrate coupled to a readout probe.

55. The method of claim 1, wherein one or more amplification probes are crosslinked to the cellular component via a functionalized hydrogel, which comprises functional groups capable of reacting with modified amplification probes.

56. The method of claim 1, wherein one or more amplification probes are crosslinked to one or more primary probes.

57. The method of claim 1, wherein one or more amplification probes are crosslinked in close proximity to one or more target analytes.

58. The method of claim 1, wherein one or more amplification probes are crosslinked in close proximity to one or more primary probes that interact with one or more analytes.

59. The method of claim 1, wherein one or more amplification probes are displaced from the primary probe by cleaving one or more amplification probes.

60. The method of claim 1, wherein one or more amplification probes bind to a contact site on at least one primary probe.

61. The method of claim 1, wherein one or more amplification probes bind to other amplification probes.

62. The method of claim 1, wherein one or more amplification probes comprise a 5' or 3' crosslinkable molecule.

63. The method of claim 62, wherein the crosslinkable molecule is a photocrosslinkable molecule.

64. The method of claim 1, wherein one or more amplification probes comprise a binding site that is reverse complementary to a binding site on one or more primary probes.

65. The method of claim 1, wherein one or more amplification probes comprise one or more readout sites, or a repeat sequence of the same readout site on a primary probe or another amplification probe.

66. The method of claim 1, wherein one or more amplification probes are separated by washing, displacement, cleavage, photocleavage, chemical reduction, chemical degradation, enzymatic digestion, an enzymatic reaction that modifies the amplification probe, or any combination thereof.

67. The method of claim 1, wherein one or more amplification probes are separated by one or more displacement probes.

68. The method of claim 67, wherein one or more displacement probes compete with the interaction between the primary probe and the amplification probe.

69. The method of claim 1, wherein one or more amplification probes are separated by binding one or more high affinity locked nucleic acid (LNA) or RNA probes.

70. The method of claim 1, wherein one or more amplification probes are separated by binding one or more displacement probes that are cross-linked to the cellular component.

71. The method of claim 1, further comprising detecting a signal generated by two amplicon interactions.

72. The method of claim 71, further comprising contacting the sample with an aptamer or bridge probe to generate a signal that indicates the physical proximity of two or more analytes.

73. The method of claim 71, further comprising contacting the sample with an aptamer or bridge probe to generate a signal that indicates the physical proximity of two or more primary probes.

74. The method of claim 1, further comprising amplifying a product from two or more primary probes.

75. The method of claim 74, wherein the product generates a signal that acts as a coincidence detector.

76. The method of claim 75, wherein the coincidence detector indicates whether the interaction between the two or more primary probes is specific or non-specific.

77. The method of claim 70, wherein one or more displacement probes comprise one or more binding sites for secondary, tertiary, and / or quaternary probe binding.

78. The method of claim 1, wherein one or more amplification probes are separated by cleavage of the amplification probe based on light.

79. The method of claim 70, further comprising a washing step wherein cleaved amplification probes are separated from the analyte.

80. The method of claim 1, wherein one or more amplification probes are separated by photocleavage of the amplification probe.

81. The method of claim 1, wherein one or more amplification probes are separated by reduction of a disulfide bridge within a secondary or tertiary probe that interacts with the amplification probe to cleave it.

82. The method of claim 1, wherein one or more amplification probes are separated by cleavage of a pH-dependent moiety within a secondary or tertiary probe that interacts with the amplification probe.

83. The method of claim 1, wherein one or more amplicon probes are separated by site-specific protease cleavage of the amplicon probe by TEV protease, ribonuclease, TALEN, zinc finger protein, lipase, or endoglycosidase.

84. The method of claim 1, wherein one or more amplicon probes are separated by electrochemical cleavage of the amplicon probe.

85. The method of claim 1, wherein one or more amplicon probes are separated by cleavage of the amplicon probe at a recognition site, or restriction endonuclease cleavage of a double stranded probe.

86. The method of claim 85, wherein the amplicon probe is cleaved by Uracil-Specific Excision Reagent (USER) enzyme.

87. The method of claim 1, wherein the amplicon probe is separated by CRISPR Cas9 modification.

88. The method of claim 1, wherein one or more amplicon probes are separated by target- specific endonuclease cleavage of the amplicon probe.

89. The method of claim 1, wherein one or more amplicon probes are separated by partial or complete removal of secondary or tertiary probes that interact with the amplicon probe.

90. The method of claim 1, wherein one or more amplicon probes are separated by stringent wash conditions of the sample.

91. The method of claim 90, wherein the stringent wash conditions comprise 30%, 40%, 50%, 55%, 60%, or 70% formamide in a buffer or aqueous solution.

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