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

Abstract

The present disclosure provides methods for the scalable amplification of the signal of an analyte in a sample for the spatial localization of the analyte in a biological sample. This disclosure sets forth methods, in addition to using the same, and other solutions to problems in the relevant field.

Description

SIGNAL AMPLIFICATION METHODS AND COMPOSITIONS FOR MOLECULARTARGET DETECTION BY ITERATIVE PROBE DEPOSITIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 524,408, filed June 30, 2023. The contents of the above-referenced application are hereby incorporated by reference in their entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH ORDEVELOPMENT

[0002] The U.S. Government has certain rights in this disclosure pursuant to Grant No NS 131408 awarded by the National Institute of Health.FIELD

[0003] The present disclosure provides methods, compositions, and kits for the scalable amplification of the signal of an analyte in a sample for the spatial localization of the analyte in a biological sample. This invention can be implemented for multiplex profiling of one or multiple ty pes of analytes such as, but not limited to, RNAs, DNAs, proteins, small molecule inhibitors, sugars, lipids, organelle, synthetic barcodes, in biological or clinical samples.BACKGROUND

[0004] The spatial profiling of cellular components, including DNA regions, proteins, and mRNAs, plays an important role in understanding cellular functions and the molecular mechanisms in health and disease. Microscopy, capable of resolving these targets in individual cells, provides crucial insights into the abundance and localization of these components.

[0005] Spatial transcriptomics has emerged as a powerful tool for understanding the spatial organization of gene expression within tissues and cells, providing valuable insights into cellular heterogeneity and tissue function (1). A major limitation for imaging-based spatial transcriptomics methods is the low signal-to-noise ratio, particularly in thick tissue samples or non-ideal tissue samples such as FFPE or human derived tissue samples.Generally, dim signals can be circumvented by using a larger number of hybridization probes per gene. Further, this approach will not work when targeting short RNAs species.

[0006] Various methods have been developed to address these challenges such as ClampFISH (2, 3). RollFISH (4), STARmap (5. 6), HyblSS (Ref 7), FISSEQ (8). hybridization chain reaction (9, 10), and branched DNA (1 1, 12). However, these methods often exhibit several deficiencies such as poor probe diffusion, non-specific signal, low detection efficiency, inability' to scale to near transcriptome-level profiling, or the necessity to extensively screen amplifier probes. Most of these approaches require enzymes which can be costly and require special storage conditions, rely on highly purified and bi-functionalized probes which can also be costly and time consuming to generate, depend on unstable reagents which can limit reproducibility and scalability, or require pre-programed probes to assemble with a defined amplification factor which does not offer a modularity to the overall method.SUMMARY

[0007] The present disclosure provides methods for the scalable amplification of the signal of an analyte in a sample for the spatial localization of the analyte in a biological sample. This disclosure sets forth methods, in addition to using the same, and other solutions to problems in the relevant field.

[0008] In some embodiments, there is provided a method for the scalable amplification of the signal of an analyte in a sample for the spatial localization of the analyte in a biological sample, comprising contacting one or more target analytes in a sample with a plurality of primary probes, wherein each probe in the plurality of primary probes interacts with at least one target analyte. In some embodiments, the method comprises contacting each of a plurality of the primary probes, each interacting wi th at least one analyte, with one or more amplifier probes. In some embodiments, the method comprises optionally, cross-linking one or more amplifier probes to a cellular component. In some embodiments, the method comprises optionally, separating each of one or more amplifier probes from its primary’ probe or from another amplifier probe. In some embodiments, the method comprises optionally, repeating any of the previous embodiments either alone or in combination. In some embodiments, the method comprises detecting one or more target analytes.

[0009] In some embodiments, the method comprises contacting a plurality of the amplifier probes each with one or more readout probes. In some embodiments, the methodcomprises imaging the sample so that the interaction of the readout probes with their target analytes is detected.

[0010] In some embodiments, the method comprises imaging the samples after contacting the amplifier probes with one or more readout probes.

[0011] In some embodiments, the method further comprises amplifying the amplifier probes by contacting the amplifier probes from a previous contacting step with anew plurality of amplifier probes.

[0012] In some embodiments, the method further comprises repeating the contacting the amplifier probes with one or more readout probes and imaging steps, each time with a new plurality of readout probes, so that the target analyte is described by a barcode, and can be differentiated from another target analyte in the sample by a difference in their barcodes. In some embodiments, the method comprises optionally, separating an amplifier probe from the primary probe or another amplifier probe after imaging the sample. In some embodiments, the method comprises optionally, separating the readout probes from the amplifier probes.

[0013] In some embodiments, there is provided a method for the scalable amplification of the signal of an analyte in a sample for the spatial localization of the analyte in a biological sample, comprising contacting one or more analytes with a primary probe in a plurality of primary probes, wherein each probe in the plurality7of primary7probes interacts with at least one target analyte. In some embodiments, the method comprises contacting each primaryprobe interacting with at least one analyte, with one or more amplifier probes. In some embodiments, the method comprises cross-linking the amplifier probe to a cellular target. In some embodiments, the method comprises separating the amplifier probes from the primary probes. In some embodiments, the method comprises optionally, repeating any of the previous steps. In some embodiments, the method comprises contacting each amplifier probe with one or more readout probes. In some embodiments, the method comprises imaging the sample after contacting the amplifier probes with one or more readout probes so that the interaction of readout probes w ith their target analytes are detected.

[0014] The methods detailed in this disclosure present a substantially more scalable, cost- effective. and modular solution. We provide a method where the amplification factor can be modularly tuned in situ, utilizes easily functionalized oligonucleotides with moderate purity, and provides a non-enzymatic solution to in situ amplification.BRIEF DESCRIPTIONS OF THE DRAWINGS

[0015] Figure 1 depicts an exemplary design of a linear amplification process. Figure 1 A illustrates an exemplary embodiment of a primary probe binding to a target molecule. Figure IB illustrates an exemplary' embodiment of an amplifier probe binding to a unique region of the primary probe. Figure 1C illustrates an exemplary embodiment of the amplifier probe binding to the sample through the reactive group. Figure ID illustrates an exemplary embodiment of subsequent amplifier binding to the primary probe. Figure IE illustrates an exemplary embodiment of multiple rounds of amplifier binding to the primary probe, binding to the sample, and unbinding from the primary- probe to allow the next amplifier to bind to the same primary’ probe site. Figure IF illustrates an exemplary embodiment of readout probes binding to the amplifiers that are bound to the sample in order to detect the target molecule.

[0016] Figure 2 depicts an exemplary design of a process without binding probes to the sample. Figure 2A illustrates an exemplary embodiment of a primary probe binding to a target molecule. Figure 2B illustrates an exemplary embodiment of amplifier probe binding to a binding region of the primary probe. Figure 2C illustrates an exemplary embodiment of the amplifier probe not binding to the sample. Figure 2D illustrates an exemplary embodiment of readout probes not binding to the sample, as depicted by the dotted line.

[0017] Figure 3 depicts an exemplary design of a pieceyvise linear amplification process. Figure 3A illustrates an exemplary embodiment of multiple amplifier probes binding to the sample. Figure 3B illustrates an exemplary embodiment of secondary amplifier probes contacting the primary- probes that are bound to the sample. Figure 3C illustrates an exemplary embodiment of binding the secondary amplifier probes to the sample. Figure 3D illustrates an exemplary embodiment in which in a subsequent round of secondary amplifier addition, sample binding, and unbinding from the initial amplifier probe, more secondary amplifiers are added. Figure 3E illustrates an exemplar}' embodiment in which during another subsequent round of secondary amplifier addition, sample binding, and unbinding from the initial amplifier probe, more secondary amplifiers are added. Figure 3F illustrates an exemplary embodiment in which each secondary amplifier probe can be bound by labelled readout probes that can be used to detect the molecular target in the sample.

[0018] Figure 4 depicts an exemplary design of a linear and exponential amplification process. Figure 4A illustrates an exemplary embodiment of multiple amplifier probes binding to the sample. Figure 4B illustrates an exemplary embodiment of secondary amplifier probes contacting the primary probes that are bound to the sample. In the example, each amplifierused for exponential amplification rounds has two binding sites for the next round of amplifiers. Figure 4C illustrates an exemplary embodiment of binding an exponential amplifier probe to the sample. In this example, the amplifiers have two binding sites for the next round of amplifiers. Figure 4D illustrates an exemplary embodiment of binding an exponential amplifier probe to the sample, with two amplifiers binding to each amplifier bound in the previous round. In this example, the amplifier probes applied in exponential amplifier round 2 can be bound with the amplifier probes applied in round 1. which in turn can be bound by the amplifier probes applied in round 2 and so forth.

[0019] Figure 5 depicts an exemplary design of an exponential amplification process following an amplification factor according to a Pell number series. Figure 5A illustrates an exemplary embodiment of a primary probe binding to a target molecule and an amplifier probe bound to the primary probe. Figure 5B illustrates an exemplary embodiment of two secondary amplifier probes binding at feature A- to the crosslinked and cleaved first amplifier at feature A+. Figure 5C illustrates an exemplary embodiment of the next round in which the same amplifier is applied to the sample as in the initial round. Figure 5D illustrates an exemplary embodiment of another round of amplification with secondary amplifier probes, which bind to the A+ sites as described in Figure 5B. Figure 5E illustrates an exemplary embodiment of the next round of amplification, in which the first amplifier is added to the sample again. Binding is similar as described in Figure 5C.

[0020] Figure 6 depicts an exemplary design of an exponential amplification process without cleavage or displacement. Figure 6A illustrates an exemplary embodiment of a primary probe binding to a target molecule and an amplifier probe bound to the primary probe. Components of the amplifier probe that are labelled in this example are a binding site R- for the primary probes, two A+ binding sites for subsequent amplifier probe binding, and a linkable feature that can bind to, for example, the sample. Figure 6B illustrates an exemplary embodiment of two subsequent amplifier probes binding to the amplifier probe that was bound to the primary probe in the previous panel. Linkable features of the amplifiers in this illustration are stably bound to the sample. Components of the amplifier probe that are labelled in this example are a binding site A- for the amplifier binding site A+ of the previous round, two R+ binding sites for subsequent amplifier probe binding, and a linkable feature. Figure 6C illustrates an exemplary' embodiment of two subsequent amplifier probes binding to the amplifier probes bound in the previous round. The amplifier probes in this example have the same components as used in round 1 shown in Figure 6A. Additional rounds of exponential amplification beyond the 3 amplification rounds shown in this exemplaryembodiment increase the number of amplifiers bound in proximity of the primary probe binding site.

[0021] Figure 7 depicts an exemplary design of amplifier probes that can be bound to the sample. Figure 7A illustrates an exemplary embodiment of a primary probe bound with an amplifier probe. Figure 7B illustrates an exemplary embodiment of an amplifier probe similar to the one described in example Figure 7A, but it shows two binding sites A+ instead of one binding site for designs that involve, for example, stronger linear amplification or non-linear amplification schemes. The cleavage or displacement feature included in this example illustration can either be a dedicated part of the probe, or partially or completely overlap with the A+ example binding site adjacent to the R- primary probe binding site for multiple purposes as a displacement, cleavage, and / or amplifier and readout probe binding site.

[0022] Figure 8 depicts an example design of a protocol for the displacement of probes using photo-crosslinking and displacement from the primary probe. Figure 8A illustrates an exemplary embodiment of a primary probe binding to a target molecule and amplifier probes binding to the primary probe. Figure 8B illustrates an exemplary embodiment of a more detailed view of the amplifier probe binding to the primary probe at site R3+ / R3-. The crossed-out circle in this example represents binding of the amplifier to the sample through the linkable feature. Figure 8C illustrates an exemplary embodiment of a displacement probe binding to the amplifier probe. In this example, binding is aided by a toehold feature T-+ on the amplifier probe, which allows binding of the displacement probe to be favoured compared to the binding of R3- to R3+ on the primary probe. Figure 8D illustrates an exemplary embodiment of displacement probes remaining attached to the amplifier probe after washing away excess displacement probes. Displacement probes remain on the amplifier probe to avoid re-binding of the amplifier probe to the primary probe. Figure 8E illustrates an exemplary embodiment of a subsequent amplification round with another amplifier probe binding to feature R3+ of the primary probe and being bound to the sample through a linkable feature (circle). The amplification cycle numbers used for the linear signal amplification are shown above the example images.

[0023] Figure 9 depicts an example design of a protocol for the displacement of probes using photo-crosslinking and displacement from the primary probe with displacers that partially bind to a second readout site on the amplifier probe. Figure 9A illustrates an exemplary embodiment of a primary probe binding to a target molecule and amplifier probes binding to the primary probe. Figure 9B illustrates an exemplary embodiment of a more detailed view of the amplifier probe binding to the primary probe at site R3+ / R3-. In thisexemplary embodiment, the amplifier probe has two A3+ amplifier binding sites, instead of a dedicated toehold sequence. The crossed-out circle in this example represents binding of the amplifier to the sample through the linkable feature. Figure 9C illustrates an exemplar}' embodiment of a displacement probe binding to the amplifier probe. In this example, binding is aided by a toehold feature that partially covers amplifier binding site A3+ on the amplifier probe, which allows binding of the displacement probe to be favoured compared to the binding of R3- to R3+ on the primary probe. In this exemplary embodiment, the toehold consists of nucleotide 1 to nucleotide n, which can for example be a total length of 10 nt, of the amplifier binding site A3+. While the displacer probe in this exemplary' embodiment can remain bound to the amplifier probe, it allows for the same amplifier probe to be used in exponential amplification rounds when no displacer probe is applied to the sample. Figure 9D illustrates an exemplary embodiment of displacement probes remaining attached to the amplifier probe after washing away excess displacement probes. Displacement probes remain on the amplifier probe to avoid re-binding of the amplifier probe to the primary probe. Figure 9E illustrates an exemplary’ embodiment of a subsequent amplification round with another amplifier probe binding to feature R3+ of the primary probe and being bound to the sample through a linkable feature (circle).

[0024] Figure 10, provides an example design of a protocol for the iterative accumulation of probes near the primary probe binding site using photo-crosslinking, amplifier cleavage, and subsequent washing for unbinding from the primary probe. Figure 10A illustrates an exemplary embodiment of a primary' probe binding to a target molecule and amplifier probes binding to the primary probe similar to Figure 7A. Figure 10B illustrates an exemplary' embodiment of a more detailed view of the amplifier probe binding to the primary probe at site R3+ / R3-. The crossed-out circle in this example represents binding of the amplifier to the sample through the linkable feature. The white rectangle illustrates an example cleavable feature of the amplifier probe. Figure 10C illustrates an exemplary embodiment of the amplifier probe that was cleaved at the cleavable feature. The portion of the amplifier that includes binding feature A3+ remains in proximity to the primary probe, as it was linked to the sample through the linkable feature illustrated by the crossed-out circle. Figure 10D illustrates an exemplary embodiment of the remaining portion of the cleaved amplifier which remains bound to example binding site R3+ through binding feature R3-. This portion of the cleaved amplifier can be removed, for example, through wash conditions suitable for the short remaining fragment. Figure 10E illustrates an exemplary embodiment of another amplifier probe binding to feature R3+ of the primary probe and being bound to the samplethrough a linkable feature (circle), which has become available through the steps described in exemplary Figures 10B-D.

[0025] Figure 11 depicts exemplary images of a linear amplification process of the fluorescence signal intensity using diazirine modified oligonucleotides of a single gene in a cell culture sample. Figure 11 A illustrates an exemplary embodiment of a fluorescent intensity signal increase over multiple rounds of linear amplification. Figure 1 IB illustrates an exemplary embodiment of a fluorescent intensity signal increase after five consecutive rounds of linear amplification. Figure 11 C illustrates an exemplary embodiment of a fluorescent intensity signal increase after eleven consecutive rounds of linear amplification.

[0026] Figure 12 depicts exemplary images of a linear amplification process of the fluorescence signal intensity using benzophenone modified oligonucleotides of a single gene in a cell culture sample. The scale bar is 20 microns. Figure 12 illustrates an exemplary embodiment of a fluorescent intensity signal increase over multiple rounds of linear amplification of benzophenone functionalized oligonucleotides.

[0027] Figure 13 illustrates an exemplary embodiment of a fluorescent intensity distribution change over multiple rounds of linear amplification of benzophenone functionalized oligonucleotides.

[0028] Figure 14 illustrates an exemplary embodiment of a combined linear and exponential amplification process of the fluorescence signal intensity of a single gene in a cell culture sample using benzophenone modified oligonucleotides. Figure 14A illustrates an exemplary embodiment of a fluorescent intensity signal increase. Labelling above exemplary images shows number of total amplification rounds. Contrast is adjusted so that images look similar despite a fluorescence increase to illustrate the similarity of fluorescent signal features such as signal spot size and signal spot number-in different amplification rounds. Figure 14B illustrates an exemplary embodiment of a fluorescent intensity signal increase over multiple rounds of amplification of benzophenone functionalized oligonucleotides. In this exemplary image, peak intensities of individual dots were plotted for each imaging round, and the fold increase of fluorescence intensity is written above the respective data points.

[0029] Figure 15 illustrates additional designs for crosslinking amplification. Figure 15 A illustrates schematics of a sacrificial layer design for amplification. Figure 15B illustrates quantification of signal of individual amplified dot in cells implementing the scheme shown in Figure 15 A. The histogram shows the intensities of the single dots in cells compared to single molecule FISH (smFISH) imaging without amplification. Figure 15C illustrates a bridge adapter design that generates signal only when two amplified balls are physicallyproximal to each other. The highlighted bridge probe with a thicken line shows the bridge adapter that bind across two amplified balls. The thicken line represents a readout probe binding site. Figure 15D illustrates images of cells amplified using the scheme shown in Figure 15C. In addition, non-specifically amplified dots in each of the amplified channels (left three panels) are not observed in the bridge adapter channel (right panel). The lower right comers of each image show zoomed-in images shown in the white box. The arrows in the insets on the lower right show regions where nonspecific dots appear in the individual amplified channel, but not in the bridge channel.

[0030] Figure 16 illustrates an exemplary embodiment of a combined linear and exponential amplification process of the fluorescence signal intensity of a single gene in a cell culture sample using benzophenone modified oligonucleotides. Figure 16A illustrates an exemplary’ embodiment of a fluorescent intensity signal increase for a small part of a recorded image to visualize individual fluorescence intensity’ peaks. Labelling above exemplary images shows number of total amplification rounds. Contrast is matched for all example images, with a lOx shorter exposure time for the rightmost two images to illustrate the increase in signal intensity. Figure 16B illustrates an exemplary embodiment of a fluorescent intensity' signal increase over multiple rounds of amplification of benzophenone functionalized oligonucleotides. In this exemplary' image, peak intensities of individual dots were plotted for each imaging round, and the fold increase of fluorescence intensity is written above the respective data points.

[0031] Figure 17 illustrates that probes can be crosslinked to the cell via CuAAC reaction. Figure 17A is a schematic diagram of DNA oligo “clicked7’ to the cell by the 3’ azide modification. Figure 17B is a schematic diagram for the CuAAC crosslinking efficiency experiment. Primary’ probes and the amplifier tested were designed as show n, and only the amplifier was crosslinked to the cell. Figure 17C, illustrates examples of the smFISH signal of 1 cycle of crosslinked amplifier on 24 Eef2 primary probes. Pre- and post- 60% formamide wash, with and without click are shown in comparison, contrasts are adjusted to the same values for each channel. Figure 17D illustrates a quantification of the signal intensity (peak value) for C. Images are Z projected for quantification.

[0032] Figure 18 illustrates how a split design can increase amplifier targeting specificity. Figure 18A is a schematic diagram of the Split probes click-crosslinked amplification method. Figure 17B illustrates an example of the Eef2 mRNA signal pre- (right, A488) and post- (left, Cy3B) 8 cycles of split probes amplification.

[0033] Figure 19 illustrates branch design enables rapid amplification with short amplifiers. Figure 19A illustrates a schematic diagram of the Branch probes click- crosslinked amplification method. Figures 19B-C, Example of Eef2 signal from 24 primary probes after 6 cycles of amplification. Figure 19B illustrates that all amplifier binding sites were designed as 13 nucleotide (nt) (13nt BS). Figure 19C illustrates that all amplifier binding sites were designed as 15 nt (15nt BS). Figure 19D illustrates quantification of the peak intensity of dots for standard smFISH, 13nt BS amplified and I5nt BS amplified. The amplification fold for 13nt BS is 19.35 (mean) and 16.64 (median); the amplification fold for 15nt BS is 39.43 (mean) and 37.36 (median). Images are Z projected for quantification.

[0034] Figure 20 illustrates that a padlock design can reduce noise by exonuclease digestion. Figure 20A illustrates a schematic diagram of the Padlock probes click-crosslinked amplification method. Figure 20B is an example of the single Ee£2 primary probe signal after 14 cycles of amplification. Figure 20C illustrates quantification of the peak intensity of dots for standard smFISH (24 primary probes) versus 1 primary probe amplified. The amplification fold for the mean is 8.41 and for the median is 7.67. Primary probes are expected to have 80% binding efficiency, therefore the amplification fold for the single probe is estimated to be ~150x.DETAILED DESCRIPTION

[0035] The following description is presented to enable one of ordinary skill in the art to make and use the disclosed subject matter and to incorporate it in the context of applications. Various modifications, as well as a variety of uses in different applications, will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to a wide range of embodiments. Thus, the present disclosure is not intended to be limited to the embodiments presented, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.DEFINITIONS

[0036] As used herein, the terms '’approximately'’ or "‘about” in reference to a number are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).

[0037] 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.

[0038] Oligonucleotides can be of various lengths. In particular embodiments, oligonucleotides can range from about 2 to about 500 nucleotides in length. In various related embodiments, oligonucleotides, single-stranded, double-stranded, and triple-stranded, can range in length from about 12 to about 20 nucleotides, from about 10 to about 60 nucleotides, from about 10 to about 90 nucleotides, from about 15 to about 30 nucleotides, from about 20 to about 120 nucleotides in length. In some embodiments, the oligonucleotide is from about 4 to about 39 nucleotides in length. In some embodiments, the oligonucleotide is at least 4 nucleotides in length. In some embodiments, the oligonucleotide is at least 5 nucleotides in length. In some embodiments, the oligonucleotide is at least 6 nucleotides in length. In some embodiments, the oligonucleotide is at least 7 nucleotides in length. In some embodiments, the oligonucleotide is at least 8 nucleotides in length. In some embodiments, the oligonucleotide is at least 9 nucleotides in length. In some embodiments, the oligonucleotide is at least 10 nucleotides in length. In some embodiments, the oligonucleotide is at least 11 nucleotides in length. In some embodiments, the oligonucleotide is at least 12 nucleotides in length. In some embodiments, the oligonucleotide is at least 15 nucleotides in length. In some embodiments, the oligonucleotide is at least 20 nucleotides in length. In some embodiments, the oligonucleotide is at least 25 nucleotides in length. In some embodiments, the oligonucleotide is at least 30 nucleotides in length. In some embodiments, the oligonucleotide is a duplex of complementary strands of at least 18 nucleotides in length. In some embodiments, the oligonucleotide is a duplex of complementary strands of at least 21 nucleotides in length.

[0039] As used herein, the term “probe” or “probes” refers to any molecules, synthetic or naturally occurring, that can attach themselves directly or indirectly to a molecular target (e.g., an mRNA sample, DNA molecules, protein molecules, RNA and DNA isoform molecules, single nucleotide polymorphism molecules, and etc.). For example, a probe can include a nucleic acid molecule, an oligonucleotide, a protein (e.g.. an antibody or an antigen binding sequence), or combinations thereof. For example, a protein probe may be connected with one or more nucleic acid molecules to form a probe that is a chimera. As disclosed herein, in some embodiments, a probe itself can produce a detectable signal. In some embodiments, a probe is connected, directly or indirectly via an intermediate molecule, with a signal moiety (e.g., a dye or fluorophore) that can produce a detectable signal.

[0040] 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, a source of interest comprises an organism, such as an animal, plant, microorganism or human. In some embodiments, a biological sample comprises biological tissue or fluid. In some embodiments, a biological sample is or comprises bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell-containing body fluids; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as a ductal lavages or bronchoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and / or excretions; and / or cells therefrom, etc. In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by methods selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g.. blood, lymph, feces etc.), etc. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and / or by adding one or more agents to) a primary' sample. For example, filtering using a semi-permeable membrane. Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary' sample to techniques such as amplification or reverse transcription of mRNA, isolation and / or purification of certain components, etc. In some embodiments, the term “sample” refers to a nucleic acid such as DNA, RNA. transcripts, or chromosomes. In some embodiments, the term “sample” refers to nucleic acid that has been extracted from the cell.

[0041] As used herein, the term “target analyte” or “analyte” refers to transcripts, RNA, DNA loci, chromosomes, DNA, exogenous DNA, proteins, peptides, lipids, glycans, cellular component, small molecules, metabolites, primary’ probe, amplifier probe, organelles and any combinations thereof.

[0042] As used herein, the term “cellular components” refers structures in a cell that regulating movement, maintain cell shape, producing proteins, or any combination thereof. In certain embodiments, the cellular components are selected from a nucleolus, a nucleus, ribosomes, vesicles, a rough endoplasmic reticulum, a golgi apparatus, a cytoskeleton, a smooth endoplasmic reticulum, mitochondrion, vacuoles, cell cytosol, lysosomes.centrosomes, cell membranes, and any combination thereof. In some embodiments, the “cellular components” refers to transcripts, RNA, DNA loci, chromosomes, DNA, proteins, peptides, lipids, glycans, small molecules, metabolites, primary probe, amplifier probe, organelles, and any combinations thereof.

[0043] As used herein, the term “primary7probe” refers to a probe that interacts with a target analyte. In certain examples, the probe is nucleic acid molecule. In certain examples, the probe is an oligonucleotide.

[0044] As used herein, the term ’‘amplifier probe” refers to a probe that interacts with a primary' probe or another amplifier probe. A “primary7amplifier” probe is an amplifier probe that interacts with a primary probe. A “secondary amplifier probe” is an amplifier probe that interacts with a primary amplifier probe or with a primary probe. A “tertiary amplifier probe” is an amplifier probe that interacts with a secondary amplifier probe or with a primary amplifier probe. A “quaternary amplifier probe” is an amplifier probe that interacts with a tertiary amplifier probe, or w ith a secondary amplifier probe. In certain embodiments, the amplifier probe is a nucleic acid sequence. In certain embodiments, the amplifier probe is an oligonucleotide. In certain embodiments, the amplifier probe has a motif that crosslinks to a cellular component.

[0045] As used herein, the term “readout probe” refers to a probe that interacts with an amplifier probe. In certain embodiments, the readout probe is a nucleic acid sequence comprising one or more fluorophores. In certain embodiments, the readout probe is an oligonucleotide.

[0046] As disclosed herein, the term “barcode” refers to a sequence signals produced by methods described herein. The barcode sequence typically is of a sufficient length and uniqueness to identity a molecular target in a single cell.

[0047] As disclosed herein, the term “cellular components” refers to a target selected from transcripts, RNA, DNA loci, chromosomes, DNA, protein, antibodies, lipids, glycans, cellular components, organelles, synapses, cell-to-cell junctions, cellular component boundaries and any combinations thereof.

[0048] As disclosed herein, the term “distribution” refers to the location of a cellular component w'ithin a cell. In certain embodiments, the term “distributions” also refers to the interactions between cellular components and a cell, between other cellular components.

[0049] As disclosed herein, the term “mapping” refers to detecting a barcode linked to a cellular component and identifying its position intracellularly or extracellularly.

[0050] As disclosed herein, the term “interacting’' or “interacts"’ refers to the binding of two or more molecules. In certain embodiments, the term “interacting"’ or “interacts” is synonymous with “bindings” or “binds.” In certain embodiments, binding-may occur by intermolecular forces, such as ionic bonds, hydrogen bonds and Van der Waals forces, between two or more molecules. In certain embodiments, binding may refer to the formation of covalent bonds between two or more molecules. In certain embodiments, “binding” may refer to the interaction of two or more molecules directly or indirectly. In certain embodiments, two or more molecules that bind indirectly have one or more molecules that interact between the two or more molecules. As example, molecule A and molecule B interact indirectly if molecule C interacts with molecule A and molecule B. but molecule A and B do not directly interact. In certain embodiments, “binding” may refer to the hybridization of two or more nucleotide sequences. In certain embodiments, “binding” may refer to a protein-nucleotide interaction. In certain embodiments, “binding” may refer to a protein-protein interaction.

[0051] As defined herein, the term “close proximity” refers to the distance between two objects wherein the first object is about 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.OVERVIEW

[0052] The present disclosure provides methods for the scalable amplification of the signal of an analyte in a sample for the spatial localization of the analyte in a biological sample. This disclosure sets forth methods, in addition to using the same, and other solutions to problems in the relevant field.

[0053] In some embodiments, there is provided a method for the scalable amplification of the signal of an analyte in a sample for the spatial localization of the analyte in a biological sample, comprising contacting one or more target analytes in a sample with a plurality of primary probes, wherein each probe in the plurality of primary probes interacts with at least one target analyte. In some embodiments, the method comprises contacting each of a plurality of the primary probes, each interacting with at least one analyte, with one or more amplifier probes. In some embodiments, the method comprises optionally, cross-linking one or more amplifier probes to a cellular component. In some embodiments, the method comprises optionally, separating each of one or more amplifier probes from its primary’ probe or from another amplifier probe. In some embodiments, the method comprises optionally,repeating any of the previous embodiments either alone or in combination. In some embodiments, the method comprises detecting one or more target analytes.

[0054] In some embodiments, the method further comprises contacting one or more amplifier probes with one or more readout probes.

[0055] In some embodiments, the method comprises detecting signals by imaging or sequencing. In some embodiments, one or more target analytes are detected by signals or absence of signals.

[0056] In some embodiments, the method comprises at least one step of cross-linking one or more amplifier probes to a cellular component.

[0057] In some embodiments, the method comprises at least one step of separating each of one or more amplifier probes from its primary probe or from another amplifier probe.

[0058] In some embodiments, the method further comprising contacting one or more amplifier probes to another amplifier probe.

[0059] In some embodiments, the method comprises imaging the samples after contacting the amplifier probes with one or more readout probes.

[0060] In some embodiments, the method further comprises amplifying the amplifier probes by contacting the amplifier probes from a previous contacting step with anew plurality of amplifier probes.

[0061] In some embodiments, the method further comprises repeating the contacting the amplifier probes with one or more readout probes and imaging steps, each time with a new plurality of readout probes, so that the target analyte is described by a barcode, and can be differentiated from another target analyte in the sample by a difference in their barcodes. In some embodiments, the method comprises optionally, separating an amplifier probe from the primary probe or another amplifier probe after imaging the sample. In some embodiments, the method comprises optionally, separating the readout probes from the amplifier probes.

[0062] In some embodiments, the method comprises single molecule resolution of target analytes. In some embodiments, the method comprises single molecule resolution of cellular components.TARGET ANALYTE and CELLUAR COMPONENT

[0063] In certain embodiments, the target analytes comprise transcripts, RNA, DNA loci, chromosomes, DNA, proteins, peptides, lipids, glycans, small molecules, metabolites, primary probe, amplifier probe, organelles, membranes, and any combinations thereof. In certain embodiments, the target analytes are obtained from bacterial cells, archaeal cells, eukaryotic cells, or a combination thereof.

[0064] In some embodiments, the target analytes comprise molecular targets that are selected from proteins, modified proteins, transcripts, RNA, DNA loci, exogenous proteins, exogenous nucleic acids, hormones, carbohydrates, small molecules, biologically active molecules, and combinations thereof. In some embodiments, the targets comprise subcellular features. In certain embodiments, the target analytes comprise RNA-DNA interactions, RNA- protein interactions, DNA-protein interactions, protein-protein interactions, or nucleic acidsmall molecule interactions.

[0065] In some embodiments, the cellular components comprise structures in a cell that regulating movement, maintain cell shape, producing proteins, or any combination thereof. In certain embodiments, the cellular components are selected from a nucleolus, a nucleus, ribosomes, vesicles, a rough endoplasmic reticulum, a golgi apparatus, a cytoskeleton, a smooth endoplasmic reticulum, mitochondrion, vacuoles, cell cytosol, lysosomes, centrosomes, cell membranes, and any combination thereof.

[0066] In some embodiments, the cellular components comprise transcripts, RNA, DNA loci, chromosomes, DNA, proteins, peptides, lipids, glycans, small molecules, metabolites, primary probe, amplifier probe, organelles, and any combinations thereof.

[0067] In some embodiments, the cellular component is a target analyte. In some embodiments, a cellular component and a target analyte are the same. In some embodiments, a cellular component and a target analyte are different. In some embodiments, the cellular component is at 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.PRIMARY PROBES

[0068] 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.

[0069] In some embodiments, the primary probe comprises oligonucleotides that are at least 5 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 6 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 7 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 8 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 9 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 10 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 11nucleotides long. In some embodiments, the primary' probe comprises oligonucleotides that are at least 12 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 13 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 14 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 15 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 16 nucleotides long. In some embodiments, the primary’ probe comprises oligonucleotides that are at least 17 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 18 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 19 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 20 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 21 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 22 nucleotides long. In some embodiments, the primary' probe comprises oligonucleotides that are at least 23 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 24 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 25 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 26 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 27 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 28 nucleotides long. In some embodiments, the primary' probe comprises oligonucleotides that are at least 29 nucleotides long. In some embodiments, the primary probe comprises oligonucleotides that are at least 30 nucleotides long. In some embodiments, the primary probes of any of the previous embodiments comprises oligonucleotides that are less than 35, 40, 45, 50. 100, 150, 200, 250, or 300 nucleotides in length.

[0070] In some embodiments, the primary' probe selectively interacts yvith a target analyte. In certain embodiments, the primary' probe is an antibody that interacts yvith a target analyte. In certain embodiments, the primary probe is an antibody-oligonucleotide conjugate, y herein the oligonucleotide interacts wit the target analyte.

[0071] In some embodiments, the primary’ probe comprises a sequence that is 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%.

[0072] In some embodiments, the primary probe comprises one or more amplifier probe binding sites. In some embodiments, the primary probe comprises two or more amplifier probe binding sites. In some embodiments, the primary probe comprises three or more amplifier probe binding sites. In some embodiments, the primary probe comprises four or more amplifier probe binding sites. In some embodiments, the primary probe comprises five or more amplifier probe binding sites. In some embodiments, the primary probe comprises six or more amplifier probe binding sites. In some embodiments, the primary probe comprises seven or more amplifier probe binding sites. In some embodiments, the primary probe comprises eight or more amplifier probe binding sites.

[0073] In some embodiments, the one or more amplifier probe binding sites are the same sequences. In some embodiments, at least one of the amplifier probe binding sites in the one or more amplifier sequences are the same. In some embodiments, the one or more amplifier probe binding sites are different from each other.

[0074] In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 5 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 6 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 7 nucleotides long. In some embodiments, the amplifier probe binding site comprises a nucleotide sequence that is at least 8 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 9 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 10 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 11 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 12 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 13 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 14 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 15 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 16 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 17 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 18 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 19 nucleotides long. In some embodiments, the amplifier probe binding sitescomprise a nucleotide sequence that is at least 20 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 21 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 22 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 23 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 24 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 25 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 26 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is at least 27 nucleotides long. In some embodiments, the amplifier probe binding sites comprise a nucleotide sequence that is less than 35, 40, 45, 50, 100, 150, 200, 250, or 300 nucleotides in length.

[0075] In some embodiments, the primary' probe comprises one or more analyte binding sites. In some embodiments, the analyte binding sites on the primary probe are the same. In some embodiments, the analyte binding sites on the primary’ probe are different. In certain embodiments, the analyte binding site comprises a sequence that is 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%.

[0076] In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 5 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 6 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 7 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 8 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 9 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 10 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 11 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 12 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 13 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 14 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 15 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotidesequence that is at least 16 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 17 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 18 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 19 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 20 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 21 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 22 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 23 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 24 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 25 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 26 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is at least 27 nucleotides long. In some embodiments, the analyte binding site comprises a nucleotide sequence that is less than 35, 40, 45, 50, 100. 150. 200, 250, or 300 nucleotides in length.AMPLIFIER PROBES

[0077] In some embodiments, the amplifier probe is a nucleic acid sequence that interacts wi th a primary probe or another amplifier probe. In some embodiments, the amplifier probe is an oligonucleotide. In some embodiments, one or more amplifier probes comprise a secondary, tertiary, and quaternary amplifier probe.

[0078] In some embodiments, the amplifier probe interacts with a primary probe, the primary probe is a protein, by interacting with a nucleotide binding site on the protein. In some embodiments, the amplifier probe interacts with a primary probe, the primary probe is an antibody. In some embodiments, the amplifier probe interacts with a primary probe, the primary’ probe is a protein-oligonucleotide conjugate, by interacting with the oligonucleotide bound to the conjugate. In some embodiments, the amplifier probe interacts with a primary probe, the primary probe is an antibody-oligonucleotide conjugate, by interacting with the oligonucleotide bound to the conjugate.

[0079] In some embodiments, wherein the amplifier probe comprises one or more primary probe binding sites. In some embodiments, one or more amplifier probes bind to a contact site on at least one primary' probe. In some embodiments, one or more amplifierprobes comprise reverse complementary binding sites to one or more primary probe binding sites. In some embodiments, one or more amplifier probes comprise one or more unique readout sites, or repeats of the same readout site on a primary probe or another amplifier.

[0080] In some embodiments, the amplifier probe comprises a primary probe binding site, a moiety that allows unbinding of the amplifier probe from the primary' probe, one or more of the same amplifier probe binding sites, one or more different amplifier probe binding sites, one or more of the same readout probe binding site, one or more different readout probe binding site, one or more of different crosslinking sites, a secondary probe binding site, a tertiary probe binding site, or any combination thereof. In certain embodiments, the secondary' probe binding site comprises a nucleotide acid sequence that binds to another amplifier probe. In certain embodiments, the tertiary probe binding site comprises a nucleotide acid sequence that binds to another amplifier probe. In certain embodiments, the primary probe comprises one or more primary' probe binding sites. In certain embodiments, the primary probe comprises one or more moieties that allow unbinding of the amplifier probe from the primary’ probe. In certain embodiments, the primary probe comprises one or more of the same amplifier probe binding sites. In certain embodiments, the primary probe comprises one or more different amplifier probe binding sites. In certain embodiments, the primary' probe comprises one or more of the same readout probe binding site. In certain embodiments, the primary probe comprises one or more different readout probe binding site. In certain embodiments, the primary probe comprises one or more of different crosslinking sites. In certain embodiments, the primary probe comprises one or secondary probe binding sites. In certain embodiments, the primary probe comprises one or more tertiary' probe binding sites.

[0081] In some embodiments, the amplifier probe is at least 5 nucleotides long. In some embodiments, the amplifier probe is at least 6 nucleotides long. In some embodiments, the amplifier probe is at least 7 nucleotides long. In some embodiments, the amplifier probe is at least 8 nucleotides long. In some embodiments, the amplifier probe is at least 9 nucleotides long. In some embodiments, the amplifier probe is at least 10 nucleotides long. In some embodiments, the amplifier probe is at least 11 nucleotides long. In some embodiments, the amplifier probe is at least 12 nucleotides long. In some embodiments, the amplifier probe is at least 13 nucleotides long. In some embodiments, the amplifier probe is at least 14 nucleotides long. In some embodiments, the amplifier probe is at least 15 nucleotides long. In some embodiments, the amplifier probe is at least 16 nucleotides long. In some embodiments, the amplifier probe is at least 17 nucleotides long. In some embodiments, the amplifier probeis at least 18 nucleotides long. In some embodiments, the amplifier probe is at least 19 nucleotides long. In some embodiments, the amplifier probe is at least 20 nucleotides long. In some embodiments, the amplifier probe is at least 21 nucleotides long. In some embodiments, the amplifier probe is at least 22 nucleotides long. In some embodiments, the amplifier probe is at least 23 nucleotides long. In some embodiments, the amplifier probe is at least 24 nucleotides long. In some embodiments, the amplifier probe is at least 25 nucleotides long. In some embodiments, the amplifier probe is at least 26 nucleotides long. In some embodiments, the amplifier probe is at least 27 nucleotides long. In some embodiments, the amplifier probe is at least 28 nucleotides long. In some embodiments, the amplifier probe is at least 29 nucleotides long. In some embodiments, the amplifier probe is at least 30 nucleotides long. In some embodiments, the amplifier probe is of any of the previous embodiments comprises nucleotides sequences that are less than 35, 40, 45, 50, 100, 150, 200, 250, or 300 nucleotides in length.

[0082] In some embodiments, the amplifier probe comprises a primary probe binding site. In some embodiments, the primary probe binding site interacts with the primary probe.

[0083] In some embodiments, the primary probe binding site comprises a sequence that is 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%.

[0084] In some embodiments, the primary probe binding site comprises a nucleotide sequence that is at least 5 nucleotides long. In some embodiments, the primary probe binding site comprises a nucleotide sequence that is at least 6 nucleotides long. In some embodiments, the primary probe binding site comprises a nucleotide sequence that is at least 7 nucleotides long. In some embodiments, the primary’ probe binding site comprises a nucleotide sequence that is at least 8 nucleotides long. In some embodiments, the primary probe binding site comprises a nucleotide sequence that is at least 9 nucleotides long. In some embodiments, the primary' probe binding site comprises a nucleotide sequence that is at least 10 nucleotides long. In some embodiments, the primary’ probe binding site comprises a nucleotide sequence that is at least 11 nucleotides long. In some embodiments, the primary probe binding site comprises a nucleotide sequence that is at least 12 nucleotides long. In some embodiments, the primary probe binding site comprises a nucleotide sequence that is at least 13 nucleotides long. In some embodiments, the primary' probe binding site comprises a nucleotide sequence that is at least 14 nucleotides long. In some embodiments, the primary probe binding site comprises a nucleotide sequence that is at least 15 nucleotides long. In some embodiments.the primary probe binding site comprises a nucleotide sequence that is at least 16 nucleotides long. In some embodiments, the primary’ probe binding site comprises a nucleotide sequence that is at least 17 nucleotides long. In some embodiments, the primary probe binding site comprises a nucleotide sequence that is at least 18 nucleotides long. In some embodiments, the primary probe binding site comprises a nucleotide sequence that is at least 19 nucleotides long. In some embodiments, the primary’ probe binding site comprises a nucleotide sequence that is at least 20 nucleotides long. In some embodiments, the primary probe binding site comprises a nucleotide sequence that is at least 21 nucleotides long. In some embodiments, the primary probe binding site comprises a nucleotide sequence that is at least 22 nucleotides long. In some embodiments, the primary' probe binding site comprises a nucleotide sequence that is at least 23 nucleotides long. In some embodiments, the primary probe binding site comprises a nucleotide sequence that is at least 24 nucleotides long. In some embodiments, the primary probe binding site comprises a nucleotide sequence that is at least 25 nucleotides long. In some embodiments, the primary' probe binding site comprises a nucleotide sequence that is at least 26 nucleotides long. In some embodiments, the primary probe binding site comprises a nucleotide sequence that is at least 27 nucleotides long. In some embodiments, the primary probe binding site comprises a nucleotide sequence that is less than 35, 40, 45, 50, 100, 150, 200, 250, or 300 nucleotides in length.

[0085] In some embodiments, the amplifier probe comprises a moiety that allows unbinding of the amplifier probe from the primary probe. In certain embodiments, the unbinding is a disruption of the interaction of the amplifier probe from the primary probe. In some embodiments, the amplifier probe comprises a moiety’ that allows unbinding of the amplifier probe from a readout probe. In certain embodiments, the unbinding is a disruption of the interaction of the amplifier probe from the readout probe.

[0086] In certain embodiments, the moiety is a displacement probe binding site. In some embodiments, the moiety comprises a sequence that is complementary to the primary' probe or amplifier 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%.

[0087] In some embodiments, the moiety comprises a nucleotide sequence that is at least 5 nucleotides long. In some embodiments, the moiety' comprises a nucleotide sequence that is at least 6 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 7 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 8 nucleotides long. In some embodiments, the moietycomprises a nucleotide sequence that is at least 9 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 10 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 1 1 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 12 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 13 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 14 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 15 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 16 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 17 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 18 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 19 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 20 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 21 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 22 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 23 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 24 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 25 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 26 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 27 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is less than 35. 40, 45, 50, 100, 150. 200, 250, or 300 nucleotides in length.

[0088] In some embodiments, the amplifier probe comprises one or more readout probe binding sites. In some embodiments, the readout probe comprises a sequence that is complementary to the amplifier 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%.

[0089] In some embodiments, the readout probe comprises a nucleotide sequence that is at least 5 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 6 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 7 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 8 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 9 nucleotideslong. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 10 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 11 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 12 nucleotides long. In some embodiments, the moiety comprises a nucleotide sequence that is at least 13 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 14 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 15 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 16 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 17 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 18 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 19 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 20 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 21 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 22 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 23 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 24 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 25 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 26 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is at least 27 nucleotides long. In some embodiments, the readout probe comprises a nucleotide sequence that is less than 35, 40, 45, 50, 100, 150, 200, 250, or 300 nucleotides in length.

[0090] In some embodiments, the amplifier probes comprise one or more of the same readout probe binding site. In certain embodiments, the same readout probe binds to different locations of the same readout probe binding sites on the amplifier probe. In some embodiments, the amplifier probe comprises one or more different readout probe binding sites. In certain embodiments, the different readout probes bind to different locations of different readout probe binding sites on the amplifier probe.

[0091] In some embodiments, the amplifier probe comprises one or more of different crosslinking sites. In certain embodiments, the crosslinking sites comprise a region of the amplifier probe that can be crosslinked to a cellular component.

[0092] In some embodiments, one or more amplifier probes bind to other amplifier probes.

[0093] In some embodiments, the amplifier probe comprises a secondary probe binding site. In certain embodiments, the secondary probe binding site comprises a region that interacts with another amplifier probe thereby linking a primary amplifier probe to a secondary amplifier probe.

[0094] In some embodiments, the amplifier probe comprises a tertiary probe binding site. In certain embodiments, the tertiary probe binding site comprises a region that interacts with another amplifier probe, linking a primary' amplifier probe interacting with a secondary' amplifier probe to a tertiary' amplifier probe.

[0095] I some embodiments, the amplifier probe comprises a quaternary probe binding site. In certain embodiments, the quaternary probe binding site comprises a region that interacts with another amplifier probe, linking a primary amplifier probe interacting wi th a secondary' amplifier probe, the secondary' amplifier probe interacting with a tertiary' probe, to a quaternary amplifier probe.

[0096] In certain embodiments, the tertiary amplifier probe interacts with the secondary amplifier probe, and the secondary probe amplifier probe interacts with the primary probe.

[0097] In certain embodiments, the quaternary' amplifier probe interacts with the tertiary' amplifier probe, and tertiary probe interacts with the secondary amplifier probe, and the secondary probe interacts with the primary probe.

[0098] In some embodiments, a quaternary amplifier probe is identical to the secondary amplifier probe.

[0099] In certain embodiments, the quaternary' amplifier probe interacts with the secondary amplifier probe.

[0100] In certain embodiments, the quaternary' amplifier probe comprises one or more binding sites for the secondary' amplifier probe, one or more binding sites for the tertiary' amplifier probe, or any combination thereof. In certain embodiments, the tertiary amplifier probe comprises one or more binding sites for the secondary amplifier probe, one or more binding sites for the quaternary amplifier probe, or any combination thereof. In certain embodiments, the secondary amplifier probe comprises one or more binding sties for the tertiary' amplifier probe, one or more binding sites for the quaternary' amplifier probe, one or more binding sites for the primary probe, or any combination thereof.

[0101] In some embodiments, the secondary’ and tertiary probe binding sites comprise a sequence that is complementary to the primary amplifier probe. In some embodiments thesequence complementarity comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100%.

[0102] In some embodiments, the secondary, tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 5 nucleotides long. In some embodiments, the secondary, tertiary, or quaternary' amplifier probes each comprise a nucleotide sequence that is at least 6 nucleotides long. In some embodiments, the secondary, tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 7 nucleotides long. In some embodiments, the secondary, tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 8 nucleotides long. In some embodiments, the secondary, tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 9 nucleotides long. In some embodiments, the secondary, tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 10 nucleotides long. In some embodiments, the secondary, tertiary, or quaternary' amplifier probes each comprise a nucleotide sequence that is at least 11 nucleotides long. In some embodiments, the secondary, tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 12 nucleotides long. In some embodiments, the tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 13 nucleotides long. In some embodiments, the secondary, tertiary', or quaternary' amplifier probes each comprise a nucleotide sequence that is at least 14 nucleotides long. In some embodiments, the secondary, tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 15 nucleotides long. In some embodiments, the secondary, tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 16 nucleotides long. In some embodiments, the secondary', tertiary, or quaternary' amplifier probes each comprise a nucleotide sequence that is at least 17 nucleotides long. In some embodiments, the secondary, tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 18 nucleotides long. In some embodiments, the secondary', tertiary, or quaternary' amplifier probes each comprise a nucleotide sequence that is at least 19 nucleotides long. In some embodiments, the secondary', tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 20 nucleotides long. In some embodiments, the secondary, tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 21 nucleotides long. In some embodiments, the secondary7, tertiary', or quaternary7amplifier probes each comprise a nucleotide sequence that is at least 22 nucleotides long. In some embodiments, the secondary7, tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 23 nucleotides long. In some embodiments, the secondary, tertiary, or quaternary amplifierprobes each comprise a nucleotide sequence that is at least 24 nucleotides long. In some embodiments, the secondary, tertiary’, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 25 nucleotides long. In some embodiments, the secondary, tertiary’, or quaternary amplifier probes each comprise a nucleotide sequence that is at least 26 nucleotides long. In some embodiments, the secondary7, tertiary, or quaternary7amplifier probes each comprise a nucleotide sequence that is at least 27 nucleotides long. In some embodiments, the secondary, tertiary, or quaternary amplifier probes each comprise a nucleotide sequence that is less than 35, 40, 45, 50, 100, 150, 200, 250, or 300 nucleotides in length.

[0103] In some embodiments, the primary probe is amplified by contacting the primary7probe with a secondary7amplifier probe. In some embodiments, the method comprises contacting the secondary amplifier probe with a tertiary amplifier probe. In some embodiments, the method comprises contacting the tertiary’ amplifier probe with a quaternary amplifier probe, wherein the quaternary amplifier probe is the same as the secondary amplifier probe.

[0104] In some embodiments, the method further comprises amplifying one or more amplifier probes by contacting one or more amplifier probes from a previous contacting step with a new plurality’ of amplifier probes. In certain embodiments, the new plurality7of amplifier probes is the same as a previous plurality of amplifier probes.CROSS-LINKING

[0105] In some embodiments, the method comprises cross-linking one or more amplifier probes to one or more cellular components. In certain embodiments, one or more amplifier probes are cross-linked to one or more cellular components. In certain embodiments, one or more amplifier probes are cross-linked to proteins in close proximity to one or more cellular components. In certain embodiments, one or more amplifier probes are cross-linked to transcripts, RNA, DNA loci, chromosomes, DNA, proteins, lipids, glycans, cellular component, organelles and any combinations thereof in close proximity7to one or more cellular components.

[0106] In some embodiments, one or more amplifier probes are crosslinked to one or more cellular components by optically dependent chemical moieties. In certain embodiments, one or more amplifier probes are crosslinked to proteins in close proximity’ to one or more cellular components by optically dependent chemical moieties. In certain embodiments, oneor more amplifier probes are crosslinked to proteins in close proximity to transcripts, RNA, DNA loci, chromosomes, DNA, proteins, lipids, glycans, cellular component, organelles and any combinations thereof to one or more cellular components by optically dependent chemical moi eties.

[0107] In some embodiments, the optically dependent chemical moieties are selected from diazirines, benzophenones, aryl-azides, or any combination thereof. In certain embodiments, optically dependent chemical moieties are photo-crosslinkers. In certain embodiments, an amplifier probe comprises one or more optically dependent chemical moieties.

[0108] In some embodiments, one or more amplifier probes are crosslinked to one or more cellular components by exposure to light at a set wavelength, a set intensity, a set duration, or any combination thereof. In some embodiments, one or more amplifier probes are crosslinked to one or more cellular components by esters. In certain embodiments, the esters are NHS-esters.

[0109] In some embodiments, one or more amplifier probes are crosslinked to one or more cellular components by cysteine reactive reagents. In some embodiments, the reactive agents are maleimides. In some embodiments, one or more amplifier probes are crosslinked to one or more cellular components using peptide coupling reagents. In some embodiments, the peptide coupling reagents are selected from carbodiimides, uronium salts, phosphonium salts, or any combination thereof.

[0110] In some embodiments, one or more amplifier probes are crosslinked to one or more cellular components by in situ carbene generation.

[0111] In some embodiments, one or more amplifier probes are crosslinked to one or more cellular components by contacting one or more cellular components with one or more rounds of crosslinkers.

[0112] In some embodiments, the crosslinkers are selected from paraformaldehyde (PF A), PEGylated bis (sulfosuccinimidyl) suberate (BSPEG), or combinations thereof.

[0113] In some embodiments, one or more amplifier probes are crosslinked to one or more cellular components by crosslinking cysteines by having thiol modified oligo. In certain embodiments, one or more amplifier probes are reacted with BM(PEG)n.

[0114] In some embodiments, one or more amplifier probes are crosslinked to one or more cellular components by amine oligo or thiol oligo. In certain embodiments, one or more amplifier probes are crossed-linked to the cellular component by reacting the lysines, cysteines, or combinations thereof with using SM(PEG)n.

[0115] In some embodiments, one or more amplifier probes are cross-linked to one or more cellular components, the one or more cellular components are functionalized with various reactive moieties on biomolecules (e.g., amines, hydroxyls, carboxylic acids, thiols, and the like), wherein the reactive moieties provide reactive handles for chemical deposition of probes. In some embodiments, the cellular components are primed to react with reactive moieties on the one or more amplifier probes.

[0116] In some embodiments, one or more amplifier probes are crosslinked to one or more cellular components by a cross metathesis reaction. In certain embodiments, the cross metathesis reaction conjugates an alkene probe and an NHS-alkene handles.

[0117] In some embodiments, one or more amplifier probes are crosslinked to one or more cellular components by via A-halosuccinimides. and an alkene amplifier probe. In certain embodiments, the crosslinking is via a Heck coupling using a palladium catalyst.

[0118] In some embodiments, one or more amplifier probes are crosslinked to one or more cellular components by small molecule substrates coupled to a readout probe. In certain embodiments, one or more amplifier probes can be read out using riboswitches or proteins.

[0119] In some embodiments, one or more amplifier probes are crosslinked one or more cellular components with a functionalized hydrogel comprising a functional group that reacts with one or more modified amplifier probes.

[0120] In some embodiments, one or more amplifier probes are crosslinked to a primary probe. In some embodiments, one or more amplifier probes are crosslinked to one or more primary probes. In some embodiments, one or more amplifier probes are crosslinked in close proximity to one or more cellular components. In some embodiments, one or more amplifier probes are crosslinked in close proximity to a primary probe interacting with a target analyte.

[0121] In some embodiments, one or more amplifier probes are crosslinked to the cellular component via click-chemistry. In some embodiments, the click-chemistry may include but is not limited to: strain-promoted azide-alkyne cycloadditions (SPAACs); tetrazine-trans- cyclooctene ligations (TCO-Tz; or inverse-electron demand Diels-Alder reactions); thiol-ene reactions; thiol-yne reactions; oxime ligations; hydrazone ligations; Diels-Alder reactions; and inverse-electron demand Diels-Alder reactions (lEDDAs).

[0122] In some embodiments, the method comprises contacting cell samples with amine groups. In some embodiments, an amine in the cells is modified with Alkyne-PEGx-NHS esters, where N is the number of monomer units. In some embodiments, an amine in the cells is modified with Azide-PEGx-NHS esters.

[0123] In certain embodiments, cell functionalization is accomplished using thiols or mercaptans as nucleophiles to make corresponding thioesters.

[0124] In certain embodiments, alkynes are present in the cells, for example, 3'-azide modified probes that can crosslink to the cell via copper(I)-catalyzed azide-alkyne cycloadditions (CuAACs).

[0125] In some embodiments, one or more amplifier probes comprise 5' or 3' cross linkable molecules. In certain embodiments, the cross-linkable molecules are photo-cross - linkable molecules.

[0126] In some embodiments, the method comprises crosslinking the probes to cellular components via treatment of hydrazides with carbonyls to form stable hydrazones.DISPLACEMENT AND CLEAVAGE

[0127] In some embodiments, the method comprises one or more displacement probes that separate one or more amplifier probes from one or more primary probe.

[0128] In some embodiments, the method comprises displacing one or more amplifier probes from one or more primary probes by cleavage of the amplifier probe.

[0129] In some embodiments, the method comprises separating one or more amplifier probes by washing, displacement, cleavage, photocleavage, chemical reduction, chemical degradation, enzymatic digestion, enzymatic reactions modifying the amplifier, or any combination thereof.

[0130] In some embodiments, the method comprises separating one or more amplifier probes by one or more displacement probes. In certain embodiments, the method comprises one or more displacement probes competing with an interaction between one or more primary probes and one or more amplifier probes.

[0131] In some embodiments, the method comprises separating one or more amplifier probes by binding a high-affinity locked nucleic acid (LNA) or RNA probe.

[0132] In some embodiments, the method comprises separating one or more amplifier probes by binding one or more displacement probes. In certain embodiments, the method comprises one or more displacement probes crosslinking to the target analyte. In some embodiments, the method comprises one or more displacement probes comprise a binding site, wherein the binding site has reverse complementarity to a sequence on one or more amplifier probes. In certain embodiments, the method comprises one or more displacement probes interacting with the primary, secondary, tertiary, or quaternary binding sites of the amplifier probe.

[0133] In certain embodiments, one or more displacement probes comprise one or more binding sites for a secondary probe, a tertiary probe, and / or a quaternary probe binding.

[0134] In some embodiments, the method comprises separating one or more amplifier probes by light-based cleavage of one or more amplifier probes.

[0135] In come embodiments, the method comprises washing steps, wherein one or more cleaved amplifier probes are separated from the analyte.

[0136] In some embodiments, the method comprises separating one or more amplifier probes by photocleavage of one or more amplifier probes.

[0137] In some embodiments, the method comprises separating one or more amplifier probes by reducing a disulfide bridge within a secondary, or a tertiary' probe interacting with an amplifier probe to cleave it.

[0138] In some embodiments, the method comprises separating one or more amplifier probes by cleaving a pH-dependent moiety within a secondary, or a tertiary' probe interacting with one or more amplifier probes.

[0139] In some embodiments, the method comprises separating one or more amplifier probes by site-specific protease cleavages of one or more amplifier probes by TEV Protease, ribonuclease, TALEN, Zinc Finger protein, lipase, or endoglycosidase.

[0140] In some embodiments, the method comprises separating one or more amplifier probes by electrochemical cleavage of one or more amplifier probes.

[0141] In some embodiments, the method comprises separating one or more amplifier probes by enzymatic cleavage of one or more amplifier probes at recognition sites, or restriction enzymes cleaving double-stranded probes. In certain embodiments, the Uracil- Specific Excision Reagent (USER) enzyme cleaves one or more amplifier probes.

[0142] In some embodiments, the method comprises separating one or more amplifier probes by CRISPR Cas9 modification.

[0143] In some embodiments, the method comprises separating one or more amplifier probes by target-specific endonuclease to cleavage of one or more amplifier probes.

[0144] In some embodiments, the method comprises separating one or more amplifier probes by binding one or more displacement probes, the one or more displacement probes crosslinking to the cellular component.

[0145] In some embodiments, the method comprises separating one or more amplifier probes a by partial or complete removal of a secondary, or a tertiary' probe interacting with one or more amplifier probes.READOUT

[0146] 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 detectably moiety. In certain embodiments, the detectably moiety is a fluorophore.

[0147] In some embodiments, the readout probe comprises an oligonucleotide with a detectable moiety.

[0148] In some embodiments, the readout probe comprises oligonucleotides that are at least 5 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 6 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 7 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 8 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 9 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 10 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 11 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 12 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 13 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 14 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 15 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 16 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 17 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 18 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 19 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 20 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 21 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 22 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 23 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 24 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 25 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 26 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 27 nucleotides long. In someembodiments, the readout probe comprises oligonucleotides that are at least 28 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 29 nucleotides long. In some embodiments, the readout probe comprises oligonucleotides that are at least 30 nucleotides long. In some embodiments, the readout probes of any of the previous embodiments comprises oligonucleotides that are less than 35, 40, 45, 50, 100 nucleotides in length.

[0149] In some embodiments, the readout probe comprises a sequence that is 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%.

[0150] In some embodiments, the readout probes comprise oligonucleotides with the same sequence. In some embodiments, the readout probes comprise oligonucleotides with different sequences.

[0151] In some embodiments, the method comprises generating signals from in situ sequencing by sequencing by ligation or sequencing by synthesis methods. In some embodiments, the signals are generated by hybridizing a sequencing primer to the primary probes, amplifier probes, or any combination thereof. In certain embodiments, a sequencing reaction generates a fluorescent signal. In some embodiments, the signals are generated by in situ sequencing of amplifier probes.

[0152] In some embodiment, the method comprises sequencing amplicons derived from the sequencing methods of the previous embodiments. In certain embodiments, the sequencing of amplicons, maps the spatial positions of one or more cellular targets, one or more target analytes, or any combination thereof. In some embodiments, the method comprises barcoding the spatial position of target analytes, cellular targets, or any combination thereof by directly capturing the amplified products or by diffusing spatial barcodes into samples to barcode the positions, generating barcoded amplicons that are extracted and sequenced in order to map the identity and position of the molecules. In some embodiments, any of the previous embodiments are performed using microscopy slide based techniques.

[0153] In some embodiments, the method comprises detecting signals generated from the interaction of two amplicons. In some embodiments, an adapter or bridge probe generates a signal when two amplified products are physically adjacent. In certain embodiments, the adapter or bridge probe is an oligonucleotide, protein, or any combination thereof. In some embodiments, one or more of the adapters generate a signal indicating the physical proximityof two or more analytes. In some embodiments, one or more of the adapters 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, the amplified products from two or more primary probes generate a signal that functions as a coincidence detector. In certain embodiments, the coincidence detector indicates a specific vs a nonspecific interaction between the primary probes. In certain embodiments, a non-specific interaction of only one of primary probe does not produce a signal.

[0154] In some embodiments, the method comprises two or more primary probes interacting with different target analytes. In some embodiments, the method comprises amplifying products from the primary probes. In certain embodiments, the amplified products from the primary probes generate a signal enhancing specificity, the signal enhancing specificity functions as a coincidence detector, the coincidence detector functions as a indicator of the physical proximity of one or more target analytes, one or more cellular components, or any combination thereof. In certain embodiments, if the target analytes are in physical proximity, the individual amplified products from one or more of the primary probes produces a signal. In certain embodiments, the proximity between combinations of target analytes such as 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 certain embodiments, the proximity distance in which a signal is generated is tuned by the length of the adaptor or bridge probe. In some embodiments, the adaptor or bridge probe is a nucleic acid. In some embodiments, the adaptor or bridge 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 the proximity’ of the primary probes generate a new analyte for further detection, amplification, or any combination thereof.FLUROPHORES

[0155] In some embodiments, the readout probe comprises a fluorophore. In certain embodiments, fluorophore is any fluorophore deemed suitable by those of skill in the arts.

[0156] In some embodiments, the fluorophores include but are not limited to fluorescein, rhodamine, Alexa Fluors, DyLight fluors, ATTO Dyes, or any analogs or derivatives thereof. In certain embodiments, the detectable moi eties include but are not limited to fluorescein and chemical derivatives of fluorescein; Eosin; Carboxyfluorescein; Fluorescein isothiocyanate(FITC); Fluorescein amidite (FAM): Erythrosine; Rose Bengal; fluorescein secreted from the bacterium Pseudomonas aeruginosa; Methylene blue; Laser dyes; Rhodamine dyes (e.g.. Rhodamine, Rhodamine 6G, Rhodamine B, Rhodamine 123, Auramine O, Sulforhodamine 101, Sulforhodamine B, and Texas Red).

[0157] In some embodiments, the fluorphores include but are not limited to ATTO dyes; Acridine dyes (e.g., Acridine orange, Acridine yellow); Alexa Fluor; 7-Amino actinomycin D; 8-Anilinonaphthalene-l -sulfonate; Auramine-rhodamine stain; Benzanthrone; 5,12- Bis(phenylethynyl) naphthacene; 9,10-Bis(phenylethynyl)anthracene; Blacklight paint; Brainbow; Calcein; Carboxyfluorescein; Carboxyfluorescein diacetate succinimidyl ester; Carboxyfluorescein succinimidyl ester; 1 -Chi oro-9, 10-bis(phenylethynyl)anthracene; 2- Chl oro-9, 10-bis(pheny lethyny l)anthracene; 2-Chloro-9.10-diphenylanthracene; Coumarin; Cyanine dyes (e.g.. Cyanine such as Cy3 and Cy5, DiOC6, SYBR Green I); DAPI, Dark quencher, Dy Light Fluor, Fluo-4, FluoProbes; Fluorone dyes (e.g., Calcein, Carboxyfluorescein, Carboxyfluorescein diacetate succinimidyl ester, Carboxyfluorescein succinimidyl ester. Eosin, Eosin B, Eosin Y, Erythrosine, Fluorescein, Fluorescein isothiocyanate. Fluorescein amidite. Indian yellow. Merbromin); Fluoro-Jade stain; Fura-2; Fura-2-acetoxymethyl ester; Green fluorescent protein, Hoechst stain, Indian yellow, Indo-1, Lucifer yellow, Luciferin, Merocyanine, Optical brightener, Oxazin dyes (e.g., Cresyl violet, Nile blue, Nile red); Perylene; Phenanthridine dyes (Ethidium bromide and Propidium iodide); Phloxine, Phycobilin, Phycoerythrin, Phycoerythrobilin, Pyranine. Rhodamine, Rhodamine 123, Rhodamine 6G, RiboGreen, RoGFP, Rubrene, SYBR Green I, (E)-Stilbene, (Z)-Stilbene, Sulforhodamine 101, Sulforhodamine B, Synapto-pHluorin, Tetraphenyl butadiene, Tetrasodium tris(bathophenanthroline disulfonate) ruthenium(II), Texas Red, TSQ. Umbelliferone, or Yellow fluorescent protein.

[0158] In some embodiments, the fluorophores include but are not limited to Alexa Fluor family of fluorescent dyes (Molecular Probes, Oregon). Alexa Fluor dyes are widely used as cell and tissue labels in fluorescence microscopy and cell biology. The excitation and emission spectra of the Alexa Fluor series cover the visible spectrum and extend into the infrared. The individual members of the family are numbered according roughly to their excitation maxima (in nm). Certain Alexa Fluor dyes are synthesized through sulfonation of coumarin, rhodamine, xanthene (such as fluorescein), and cyanine dyes. In some embodiments, sulfonation makes Alexa Fluor dyes negatively charged and hydrophilic. In some embodiments, Alexa Fluor dyes are more stable, brighter, and less pH-sensitive than common dyes (e.g. fluorescein, rhodamine) of comparable excitation and emission, and tosome extent the newer cyanine 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.

[0159] In some embodiments, the fluorophores comprise 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 Dy Light-350, Dy Light-405.Dy Light-488, DyLight-549, DyLight-594, DyLight-633, Dy Light-649, Dy Light-680, DyLight-750, or DyLight-800.

[0160] In some embodiments, the fluorophore comprises a nanomaterial. 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 a gold nanoparticle. In some embodiments, the fluorophore is a gold nanoparticle. In some embodiments, the fluorophore comprises a gold nanoparticle.IMAGING THE SAMPLE

[0161] In some embodiments, the method comprises imaging the readout probes. In some embodiments, the method comprises imaging the barcodes. As understood by a person having ordinary skill in the art, different technologies can be used for the imaging steps.

[0162] In some embodiments, the imaging methods comprise but are not limited to epifluorescence microscopy, confocal microscopy, the different types of super-resolution microscopy (PALM / STORM, SSIM / GSD / STED), and light sheet microscopy (SPIM and etc.).

[0163] In some embodiments, the imaging methods comprise exemplary super resolution technologies include, but are not limited to I5M and 4Pi-microscopy, Stimulated Emission Depletion microscopy (STEDM), Ground State Depletion microscopy (GSDM), Spatially Structured Illumination microscopy (SSIM), Photo- Activated Localization Microscopy (PALM), Reversible Saturable Optically Linear Fluorescent Transition (RESOLFT), Total Internal Reflection Fluorescence Microscope (TIRFM). Fluorescence-PALM (FPALM), Stochastical Optical Reconstruction Microscopy (STORM), Fluorescence Imaging with One- Nanometer Accuracy (FIONA), and combinations thereof. For examples: Chi, 2009 “Superresolution microscopy: breaking the limits,’' Nature Methods 6(1): 15-18; Blow 2008, “Newways to see a smaller world,” Nature 456:825-828; Hell, et al, 2007, "Tar-Field Optical Nanoscopy,” Science 316: 1153; R. Heintzmann and G. Ficz, 2006. ‘"Breaking the resolution limit 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 I5M and 4Pi-microscopy,” 222(2): 105-1 17; and Wells, 2004, “Man the Nanoscopes.” JCB 164(3):337-340.

[0164] In some embodiments, electron microscopes (EM) are used for imaging.

[0165] In some embodiments, an imaging step detects a target. In some embodiments, an imaging step localizes a target. In some embodiments, an imaging step provides three- dimensional spatial information of a target. In some embodiments, an imaging step quantifies a target. By using multiple contacting and imaging steps, provided methods are capable of providing spatial and / or quantitative information for a large number of targets in surprisingly high throughput. For example, when using F delectably different types of labels, spatial and / or quantitative information of up to FN targets can be obtained after N contacting and imaging steps.

[0166] Certain techniques for imaging are known in the art. See, for example. International PCT Patent Application No. PCT / US2014 / 036258, filed April 30, 2014 and titled MULTIPLEX LABELING OF MOLECULES BY SEQUENTIAL HYBRIDIZATION BARCODING, the entire contents of which are herein incorporated by reference in its entirety for all purposes.

[0167] In some embodiments, the method comprises analyzing cell size and shape, markers, immunofluorescence measurements, or any combinations thereof.

[0168] In some embodiments, signals are detected by imaging or sequencing. In some embodiments, the samples are imaged after contacting one or more amplifier probes with one or more readout probes.BARCODING THE TARGETS

[0169] In some embodiments, the method comprises repeating the contacting the amplifier probes with one or more readout probes and imaging steps, each time with a new plurality of readout probes, so that the target analyte is described by a barcode, and can be differentiated from another target analyte in the sample by a difference in their barcodes.

[0170] In some embodiments, the targets that are selected from proteins, modified proteins, transcripts, RNA. DNA loci, exogenous proteins, exogenous nucleic acids, hormones, carbohydrates, small molecules, biologically active molecules, and combinations thereof. In some embodiments, the targets comprise subcellular features. For example, the nuclear lamin can be one set of barcodes, and the nucleolus can be targeted with another set of barcodes. This allows each sample can be analyzed with a combination of barcodes on different subcellular compartments. In some embodiments, the method compnses barcoding targets, wherein the targets are different.

[0171] In some embodiments, the method comprises fluorescence detection. In some embodiments, the method comprises fluorescence detection or other methods of detection. In some embodiments, the method comprises sequential hybridization to detect target analytes.

[0172] In some embodiments, the probes are used in a method to barcode one or more molecular targets. See, for example, International PCT Patent Application No.PCT / US2014 / 036258, filed April 30, 2014 and titled MULTIPLEX LABELING OF MOLECULES BY SEQUENTIAL HYBRIDIZATION BARCODING, the entire contents of which are herein incorporated by reference in its entirety for all purposes.

[0173] In some embodiments, the probes are used in a method for linked amplification tethered with exponential radiance (LANTERN). See, for example, International Patent Application No. PCT / US2022 / 021826, FILED March 24, 2022, and titled LINKED AMPLIFICATION TETHERED WITH EXPONENTIAL RADIANCE, the entire contents of which are herein incorporated by reference in its entirety for all purposes.

[0174] In some embodiments, the probes are used in a method for ClampFISH. See, for example, ClampFISH detects individual nucleic acid molecules using click chemistry -based amplification, Rouhanifard S.H. et al.. Nature Biotechnology 37: 84-89 (2019), the entire contents of which are herein incorporated by reference in its entirety for all purposes.

[0175] In some embodiments, the method comprises readout probes that are selected from proteins, modified proteins, RNA, oligonucleotides, antibodies, antibody fragments, and combinations thereof.

[0176] In some embodiments, the method comprises contacting each sample in the one or more samples with a first plurality of readout probes, so that the probes interact with one or more targets. In some embodiments, the method comprises imaging the sample after the first contacting step so that interaction of the readout probes with their targets is detected.

[0177] In some embodiments, the method comprises a contacting step that differs from another contacting step in the labelling of at least one of the targets.

[0178] In some embodiments, the method comprises a contacting step wherein each detectably labelled probe in the first plurality of probes is labelled with a detectably moiety.

[0179] In some embodiments, the method comprises a contacting step wherein each detectably labelled probe comprises a detectable moiety and at least one contacting step differs from another contacting step by having a different detectable moiety for each target.

[0180] In some embodiments, the method comprises a contacting step wherein at least two different readout probes that interact with a first target and wherein at least two different readout probes interact with a second target.

[0181] In some embodiments, the readout probes comprise labels selected from two, three, or four different labels.

[0182] In some embodiments, the barcode for the target in the sample comprises a signal that is amplified. In certain embodiments, the barcode for the target in a sample comprises a signal that is amplified by rolling circle, padlock, branched DNA, ClampFISH, LANTERN, or any combination thereof.

[0183] In some embodiments, the method comprises using readout probes wherein each detectably labelled probe comprises the same detectable moiety and the same sequence.

[0184] In some embodiments, the method comprises readout probes wherein each readout probes interacts with its target through one or more intermediate probes each of which is hybridized to the target.

[0185] In some embodiments, the method comprises repeating the contacting and imaging steps, each time with a new plurality of readout probes so that a target in the sample is described by a barcode, and can be differentiated from another target in the sample by a difference in their barcodes.

[0186] In some embodiments, the method comprises an error correction round. See, for example. International Patent Application No. PCT / US2017 / 044994, FILED August 0, 2017, and titled SEQUENTIAL PROBING OF MOLECULAR TARGETS BASED ON PSEUDOCOLOR BARCODES WITH EMBEDDED ERROR CORRECTION MECHANISM, the entire contents of which are herein incorporated by reference in its entirety for all purposes.

[0187] In some embodiments, the method comprises an error correction round performed by selecting from block codes such as Hamming codes, Reed-Solomon codes, Golay codes, or any combination thereof.

[0188] In some embodiments, the method of any of the previous embodiments further comprises an error correction step. In certain embodiments, the error correction stepcomprises performing additional rounds of contacting and imaging prior or in between or after steps (i)-(v).REMOVING PROBES

[0189] In some embodiments, the method comprises a step of removing the readout probes after one or more imaging steps. In some embodiments, the step of removing the readout probes comprises contacting the plurality of readout probes with an enzyme that digests a readout probes. In some embodiments, the step of removing comprises contacting the plurality of readout probes with a DNase, contacting the plurality of readout probes with an RNase, photobleaching, strand displacement, formamide wash, heat denaturation, or combinations thereof. In some embodiments, the step of removing comprises photobleaching to remove the readout probes.

[0190] In some embodiments, the method comprises removing readout probes by using stripping reagents, wash buffers, photobleaching, chemical bleaching, and any combinations thereof.

[0191] In some embodiments, the amplifier probe is separated by stringent wash conditions of the sample. In some embodiments, the stringent wash conditions comprise 30%, 40%, 50%, 55%, 60%, or 70% formamide in a buffered or aqueous solution.

[0192] In some embodiments, the method comprises clearing the sample. In some embodiments, the sample is cleared by CLARITY.

[0193] 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 and titled MULTIPLEX LABELING OF MOLECULES BY SEQUENTIAL HYBRIDIZATION BARCODING, the entire contents of which are herein incorporated by reference in its entirety’ for all purposes.WASHES

[0194] In some embodiments, the method of any of the preceding embodiments, comprises optionally washing the sample after each step. In certain embodiments, the sample is washed with a buffer that removes non-specific hybridization reactions. In certain embodiments, formamide is used in the wash step. In certain embodiments, the wash buffer is stringent. In certain embodiments, the wash buffer comprises 10% formamide, 2xSSC, and 0. 1% triton X-lOOs.

[0195] Having described the embodiments in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

[0196] The following non-limiting methods and examples are provided to further illustrate the embodiments disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the methods and examples that follow represent approaches that have been found to function well in practice, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the embodiments.EXAMPLES

[0197] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.EXAMPLE 1

[0198] This example, as illustrated in Figure 1, depicts an exemplary design of a linear amplification process.

[0199] Figure 1A illustrates an exemplary embodiment of a primary probe binding to a target molecule. In this example, a ssDNA primary probe binds to an RNA target at a site that is defined by a reverse complement region of the primary7probe.

[0200] Figure IB illustrates an exemplary embodiment of amplifier probe binding to a unique region of the primary probe. In this example, a ssDNA amplifier probe binds to a specific region of the ssDNA primary probe at a site where both probes have reverse complement sequences from each other. The amplifier probe is functionalized with a reactive group.

[0201] Figure 1C illustrates an exemplary embodiment of the amplifier probe binding to the sample through the reactive group. In this example, the amplifier group was modified with a photoreactive group that can be crosslinked by UV-light excitation to nearby reactivegroups in the sample. Following the binding, the amplifier probe is either cleaved and partially washed away or displaced from the primary probe so that the initial primary probe binding site for another amplifier is freed up again.

[0202] Figure ID illustrates an exemplary embodiment of subsequent amplifier binding to the primary probe. In this example, the amplifier binds at the same, freed-up location where the previous amplifier was bound.

[0203] Figure IE illustrates an exemplary embodiment of multiple rounds of amplifier binding to the primary probe, binding to the sample, and unbinding from the primary probe to allow the next amplifier to bind to the same primary' probe site. With each round the number of amplifiers in close proximity to the target molecule can increase by one. The procedure is repeated until a sufficient number of amplifiers for detection of the target molecule is accumulated in the sample.

[0204] Figure IF illustrates an exemplary embodiment of readout probes binding to the amplifiers that are bound to the sample in order to detect the target molecule. In this example, readout probes are ssDNA probes that can bind to the amplifier probes at a specific site at which both probes are reverse complement to each other.EXAMPLE 2

[0205] This example, as illustrated in Figure 2, depicts an exemplary design of a process without binding probes to the sample. Without repeated binding of amplification probes to the sample, no signal amplification can take place with the method described here.

[0206] Figure 2A illustrates an exemplary' embodiment of a primary probe binding to a target molecule. In this example, a ssDNA primary' probe binds to an RNA target at a site that is defined by a reverse complement region of the primary probe. It is the same example setting as described for Figure 1A.

[0207] Figure 2B illustrates an exemplary^ embodiment of an amplifier probe binding to a binding region of the primary' probe. In this example, a ssDNA amplifier probe binds to a specific region of the ssDNA primary probe at a site where both probes have reverse complement sequences from each other. The amplifier probe is functionalized with a reactive group. It is the same example seting as described for Figure IB.

[0208] Figure 2C illustrates an exemplary' embodiment of the amplifier probe not binding to the sample. As the amplifier probe is not linked to the sample, the example cleavage shown here results in amplifier probe removal from the sample.

[0209] Figure 2D illustrates an exemplary embodiment of readout probes not binding to the sample, as depicted by the dotted line. There are no amplifiers that are bound to the sample, and there are no available binding sites for the readout probes, so it is not possible to detect the target molecule.EXAMPLE 3

[0210] This example, as illustrated in Figure 3, depicts an exemplary design of a piecewise linear amplification process.

[0211] Figure 3A illustrates an exemplary embodiment of multiple amplifier probes binding to the sample. Following the binding, each amplifier probe was either cleaved and partially washed away or displaced from the primary probe so that the initial primary probe binding site for another amplifier is freed up again. Multiple rounds of adding amplifier rounds results in the presence of multiple amplifier rounds near the primary probe and molecular target of the probes. The procedure is repeated until a sufficient number of amplifiers for detection of the target molecule is accumulated in the sample.

[0212] Figure 3B illustrates an exemplary embodiment of secondary amplifier probes contacting the primary probes that are bound to the sample. In this example, the secondary amplifier probes have a region reverse complementary to the initially bound amplifier probes. The number of secondary amplifier probes that can bind to the sample depends on the number of initial amplifier probes present in the sample. In this exemplary embodiment, the number of secondary amplifiers that can bind to the initial amplifier probes is 6.

[0213] Figure 3C illustrates an exemplary7embodiment of binding the secondary7amplifier probes to the sample. This can be induced in the same or different ways compared to binding the initial amplifier probes. In this exemplary7embodiment, 6 secondary amplifier probes are bound to the sample.

[0214] Figure 3D illustrates an exemplary7embodiment in which in a subsequent round of secondary7amplifier addition, sample binding, and unbinding from the initial amplifier probe, 6 more secondary7amplifiers are added.

[0215] Figure 3E illustrates an exemplary embodiment in which during another subsequent round of secondary7amplifier addition, sample binding, and unbinding from the initial amplifier probe, 6 more secondary7amplifiers are added. The exemplary7process can be repeated until a sufficient number of secondary amplifiers for the molecular target detection is present.

[0216] Figure 3F illustrates an exemplary embodiment in which each secondary amplifier probe can be bound by readout probes that can be used to detect the molecular target in the sample. In this exemplary embodiment, the readout probes can be fluorophore- conjugated ssDNA oligo sequences.EXAMPLE 4

[0217] This example, as illustrated in Figure 4, depicts an exemplary design of a combined linear and exponential amplification process.

[0218] Figure 4A illustrates an exemplary embodiment of multiple amplifier probes binding to the sample. After binding, each amplifier probe was either cleaved and partially washed away or displaced from the primary probe so that another amplifier can bind to the primary probe. Multiple rounds of adding amplifier probes results in the presence of multiple amplifier probes near the primary probe and molecular target of the probes. The procedure is repeated until a sufficient number of amplifiers for detection of the target molecule is accumulated in the sample.

[0219] Figure 4B illustrates an exemplary embodiment of secondary amplifier probes contacting the primary amplifier probes that are bound to the sample. In this example, the secondary amplifier probes have a region reverse complementary to the initially bound amplifier probes. The number of secondary amplifier probes that can bind to the sample depends on the number of initial amplifier probes present in the sample. In this exemplary embodiment, the number of secondary amplifiers that can bind to the initial amplifier probes is 6. Only a single step with 6 amplifiers is shown in this example. As described before the procedure with binding, cleavage or displacement and binding of additional amplifier probes can be repeated until a sufficient number of amplifiers for detection of the target molecule is accumulated in the sample. For an exponential amplification in the subsequent steps of this example, each of the amplifier probes bound here has two binding sites for amplifier probes in the next amplification round.

[0220] Figure 4C illustrates an exemplary embodiment of binding exponential amplifier probes to the sample. In this exemplary embodiment, 12 tertiary amplifier probes are bound to the 6 amplifier probes that were bound to the sample in the previous step. In this example, no displacement or cleavage or the probes takes place. Each amplifier is bound to the sample, and has two binding sites available for subsequent amplifier probe binding.

[0221] Figure 4D illustrates an exemplary embodiment of binding exponential amplifier probes to the sample.EXAMPLE 5

[0222] This example, as illustrated in Figure 5, depicts an exemplary design of an exponential amplification process following an amplification factor according to a Pell number series.

[0223] Figure 5 A illustrates an exemplary embodiment of a primary probe binding to a target molecule and an amplifier probe bound to the primary probe. In this example, a ssDNA primary probe binds to an RNA target at a site that is defined by a reverse complement region of the primary probe. The primary probe further has a binding feature R+ for an amplifier probe, which can bind the primary probe through the reverse complementary feature R-. Dotted lines at the ends of the primary probe indicate that A pically, each primary’ probe contains multiple unique amplifier binding sites, which are omitted for clarity in this exemplary figure. The first amplifier has a feature that allows binding to the sample (circle), and a cleavable feature (square), and two binding sites for the second amplifier (A+). In total, in the step of the method labelled here as round 1. there are a total of two binding features Anin the sample.

[0224] Figure 5B illustrates an exemplary embodiment of two secondary amplifier probes binding at feature A- to the crosslinked and cleaved first amplifier at feature A+. After crosslinking and cleaving the probe, only the cross-linkable portion of the first amplifier, which includes the binding sites for the second amplifier, remains in the sample. Note that the position of the crosslinked probes is shifted in the illustration for clarity' purposes. Elements that were present before this round are illustrated in light grey for clarity'. The secondary amplifier has two binding features R+, which is designed to be identical to the binding feature R+ on the primary probe.

[0225] Figure 5C illustrates an exemplary embodiment of the next round in which the same amplifier is applied to the sample as in the initial round. The amplifier binds the secondary' amplifier at feature R+ through its binding feature R-. Again, the first amplifier has two binding features A+. The newly added number of binding features A+ in this round of amplification is 10. The total number of available binding features A+ in the sample is now 12.

[0226] Figure 5D illustrates an exemplary' embodiment of another round of amplification with secondary amplifier probes, which bind to the A+ sites as described in figure 5B.

[0227] Figure 5E illustrates an exemplary embodiment of the next round of amplification, in which the first amplifier is added to the sample again. Binding is similar as described infigure 5C. The newly added number of binding features A+ in this round of amplification is 58. The total number of available binding features A+ in the sample is now 70. The process can be repeated through additional rounds until a sufficient amount of features A+ are bound in the sample. In this example, feature A+ can be bound by fluorophore-conjugated readout probes. Alternatively, it is also possible to bind the readout probe to any other amplifier probe element shown in this example.EXAMPLE 6

[0228] This example, as illustrated in Figure 6, depicts an exemplary design of an exponential amplification process.

[0229] Figure 6A illustrates an exemplary embodiment of a primary probe binding to a target molecule and an amplifier probe bound to the primary probe. In this example, a ssDNA primary probe binds to an RNA target at a site that is defined by a reverse complement region of the primary7probe. The primary probe further has a binding feature R+ for an amplifier probe, which can bind the primary probe through the reverse complementary feature R-. Dotted lines at the ends of the primary probe indicate that typically, each primary probe contains multiple unique amplifier binding sites, which are omitted for clarity in this exemplary' figure. The first amplifier has a feature that allow s binding to the sample (circle), and two binding sites for the second amplifier (A+). In total, in the step of the method labelled here as round 1. there are a total of two binding features A+ in the sample.

[0230] Figure 6B illustrates an exemplary embodiment of two secondary amplifier probes binding at feature A- to the crosslinked first amplifier at feature A+. The secondary' amplifier has two binding features R+. w hich can be identical to the binding feature R+ of the primary probe, for an amplifier probe. After crosslinking the secondary amplifiers, in this example, no cleavage of displacement of amplifier probes takes place. In total, in the step of the example method labelled here as round 2, a total of four binding features R+ are added to the sample.

[0231] Figure 6C illustrates an exemplary embodiment of four amplifier probes, which can be identical to the amplifier probes used in round 1. binding at feature R- to the crosslinked first amplifier at feature R+. The amplifier has two binding features A+. After crosslinking the secondary amplifiers, in this example, no cleavage or displacement of amplifier probes takes place. In total, in the step of the example method labelled here as round 3, a total of 8 binding features A+ are added to the sample. The process is repeated until a sufficient number of amplifiers for detection of the target molecule is accumulated in the sample.EXAMPLE 7

[0232] This example, as illustrated in Figure 7, depicts an exemplary design of amplifier probes that can be bound to the sample.

[0233] Figure 7A illustrates an exemplary embodiment of a primary' probe bound with an amplifier probe. Details on an exemplary design of an amplifier probe are given. Only one amplifier is shown here, with potentially multiple of the same or different amplifiers binding to primary probes. The amplifier probe consists of a primary probe binding site (R-), w hich is a reverse complement to an amplifier probe binding site on the primary probe (R+). A feature that allow s cleavage or probe displacement (rectangle) is part of the amplifier and allows freeing up the primary probe site after crosslinking to complete one cycle of amplification. The cleavage site can also be placed within the primary probe binding site R- or other features of the amplifier probe adjacent to the primary' probe binding site R-. As a displacement site, R- can be a toehold sequence for a displacer strand that is reverse complementary’ to the amplifier probe. Other features of the amplifier adjacent to the primary probe binding site R- can be an additional toehold feature for effective displacer strand binding. The amplifier further features an example binding site A+, which is bound by other amplifier probes or by readout probes that allow' detection of the signal, for example by conjugating fluorescent dyes to the readout probes.

[0234] Figure 7B illustrates an exemplary embodiment of an amplifier probe similar to the one described in example Figure 7A, but it shows two binding sites A+ instead of one for designs that involve, for example, stronger linear amplification or non-linear amplification schemes.EXAMPLE 8

[0235] This example, as illustrated in Figure 8, depicts an example design of a protocol for the displacement of probes using photo-crosslinking and displacement from the primary’ probe.

[0236] Figure 8A illustrates an exemplary embodiment of a primary probe binding to a target molecule and amplifier probes binding to the primary' probe. In this example, the primary' probe has four binding sites R1+, R2+, R3+ and R4+, which can be bound by unique amplifier probes through respective sites R1-, R2-, R3- and R4-. The dotted rectangle indicates part of the primary probe that follows panels Figures 8 B-D. The white circle at theend of the amplifier probes illustrates a feature that can be bound, for example crosslinked by UV-light, to the sample.

[0237] Figure 8B illustrates an exemplary embodiment of a more detailed view of the amplifier probe binding to the primary probe at site R3+ / R3-. Besides the binding site R3-, the example amplifier has a toehold sequence binding site T+ and an amplifier binding site A3+. The crossed-out circle in this example represents binding of the amplifier to the sample through the linkable feature.

[0238] Figure 8C illustrates an exemplary embodiment of a displacement probe binding to the amplifier probe. In this example, binding is aided by a toehold feature T+ on the amplifier probe, which allows binding of the displacement probe R3+T- to be favoured compared to the binding of the amplifier at binding site R3- to the primary probe at R3+.

[0239] Figure 8D illustrates an exemplary embodiment of a displacement probe remaining attached to the amplifier probe after unbound displacer probes are washed or otherwise removed from the sample. During this step, the previously bound amplifier probe cannot bind to the primary probe at R3+ / R3- again because the bound displacement probe is blocking the binding site. The R3+ site of the primary probe therefore remains available for other amplifier probes in subsequent binding round.

[0240] Figure 8E illustrates an exemplary embodiment of another amplifier probe binding to feature R3+ of the primary probe and being bound to the sample through a linkable feature (crossed-out circle).EXAMPLE 9

[0241] This example, as illustrated in Figure 9, depicts an example design of a protocol for the displacement of probes using photo-crosslinking and displacement from the primary probe.

[0242] Figure 9A illustrates an exemplary embodiment of a primary probe binding to a target molecule and amplifier probes binding to the primary probe. In this example, the primary probe has four binding sites R1+, R2+, R3+ and R4+, which can be bound by unique amplifier probes through respective sites R1-. R2-, R3- and R4-. The dotted rectangle indicates part of the primary probe that follow s panels Figures 9 B-D. The white circle at the end of the amplifier probes illustrates a feature that can be bound, for example crosslinked by UV-light, to the sample.

[0243] Figure 9B illustrates an exemplary embodiment of a more detailed view of the amplifier probe binding to the primary' probe at site R3+ / R3-. Besides the binding site R3-,the example amplifier has two d amplifier binding sites A3+. The crossed-out circle in this example represents binding of the amplifier to the sample through the linkable feature.

[0244] Figure 9C illustrates an exemplary embodiment of a displacement probe binding to the amplifier probe. In this example, binding is aided by a toehold feature A3(l-n)-, which is the first n nucleotides of the A3- sequence that can bind to the A3+ sequence on the amplifier probe. The length is for example 10 nt, so that the displacement probe in this example has a total length of 25nt. The toehold sequence allows binding of the displacement probe R3+ A3(l-n)- to be favoured compared to the binding of the amplifier at binding site R3- to the primary probe at R3+. In addition, the R3+ A3(l-n)- displacer allows amplifier probes with two feature sites A3+ to be used flexibly also for exponential amplification rounds without displacement.

[0245] Figure 9D illustrates an exemplary embodiment of a displacement probe remaining attached to the amplifier probe after unbound displacer probes are washed or otherwise removed from the sample. During this step, the previously bound amplifier probe cannot bind to the primary probe at R3+ / R3- again because the bound displacement probe is blocking the binding site. The R3+ site of the primary probe therefore remains available for other amplifier probes in subsequent binding round.

[0246] Figure 9E illustrates an exemplary7embodiment of another amplifier probe binding to feature R3+ of the primary probe and being bound to the sample through a linkable feature (crossed-out circle).EXAMPLE 10

[0247] This example, as illustrated in Figure 10, provides an example design of a protocol for the displacement of probes using photo-crosslinking and amplifier cleavage for unbinding from the primary probe.

[0248] Figure 10A illustrates an exemplary embodiment of a primary7probe binding to a target molecule and amplifier probes binding to the primary probe similar to Figure 9A. In this example, the primary probe has four binding sites R1+, R2+, R3+ and R4+, which can be bound by unique amplifier probes through respective sites R1-. R2-, R3- and R4-. . The white circle at the end of the amplifier probes illustrate a feature that can be bound, for example crosslinked by UV -light, to the sample.

[0249] Figure 10B illustrates an exemplary embodiment of a more detailed view of the amplifier probe binding to the primary probe at site R3+ / R3-. The crossed-out circle in thisexample represents binding of the amplifier to the sample through the linkable feature. The white rectangle illustrates an example cleavable feature of the amplifier probe.

[0250] Figure 10C illustrates an exemplary embodiment of the amplifier probe that was cleaved at the cleavable feature. The portion of the amplifier that includes binding feature A+ remains in proximity to the primary probe, as it was linked to the sample through the linkable feature illustrated by the crossed-out circle.

[0251] Figure 10D illustrates an exemplary embodiment of the remaining portion of the cleaved amplifier which remains bound to example binding site R3+ through binding feature R3-. This portion of the cleaved amplifier can be removed, for example, through wash conditions suitable for the short remaining fragment.

[0252] Figure 10E illustrates an exemplary embodiment of another amplifier probe binding to feature R3+ of the primary probe and being bound to the sample through a linkable feature (circle), which has become available through the steps described in Figures 10B-10D.EXAMPLE 11

[0253] This example, as illustrated in Figure 11, depicts exemplary images of a linear amplification process of the fluorescence signal intensity using diazirine modified oligonucleotides of a single gene in a cell culture sample.

[0254] Approximately 100000 NIH3T3 cells were seeded onto a 24x60mm #1.5 glass coverslips. Cells were fixed with 4% PFA in lx PBS at room temperature for 10 min. After washing with lx PBS, samples were stored in 70% EtOH at -20C for permeabilization overnight. Samples were dried with nitrogen gas and a custom flow cell was attached to the glass coverslip. After rinsing with lx PBS, 7.5 mM BS-PEG5 in lx PBS was added for 30 min at room temperature. Samples were incubated with 100 mM / V-(Propionyloxy) succinimide in lx PBS two times for 30 min at room temperature. Samples were rinsed with 40% wash buffer (40% formamide, 2x SSC, 0.1% Triton-XlOO) three times. Primary probes were hybridized with a 40% hybridization buffer (40% formamide, 0.1 mg / mL yeast tRNA, 2X SSC, 10% 500kDa Dextran Sulfate) containing 5 nM / oligo at 37 C for 12 hours. After primary probe hybridization, the sample was rinsed with a 40% wash buffer 3 times and incubated at 37 C once. Subsequently, the sample was rinsed with 2X SSC solution. Then, 100 nM / oligo amplifier probes were hybridized to the sample in a 10% hybridization buffer (10% formamide, 2X SSC, 10% 6-10kDa Dextran Sulfate, 0.1% Triton-XlOO) at room temp for 30 min. The sample was rinsed with 10% wash buffer (Wash buffer: 10% formamide, 2XSSC, 0.1% TritonX-100) and lx PBS, then illuminated using a 365 nm UV lamp for 45 min. Next, samples were rinsed with 2x SSC and incubated with 1 uM of displacer strand in a 15% hybridization buffer at room temperature for 15 min. Post displacement, the sample was rinsed with a 20% wash buffer (20% formamide, 2X SSC, 0.1% TritonX-100). This process of amplifier hybridization, crosslinking, and displacement was repeated multiple times to obtain sufficient signal amplification. Samples ready to image were hybridized with a 10% hybridization buffer containing 100 nM / oligo readouts at room temperature for 15 min. Samples were stained with 3 ug / mL DAPI in 2X SSC for 1 min, then rinsed with 2X SSC twice. Anti-bleaching 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) w as applied and samples were imaged using a 63x, 1.4 NA Leica objective and a spinning disk confocal microscope (Andor Dragonfly).

[0255] Figure 11 A illustrates an exemplary embodiment of a fluorescent intensity signal increase over multiple rounds of linear amplification.

[0256] Figure 1 IB illustrates an exemplary embodiment of a fluorescent intensity signal increase after five consecutive rounds of linear amplification.

[0257] Figure 11C illustrates an exemplary embodiment of a fluorescent intensity signal increase after eleven consecutive rounds of linear amplification.Example 12

[0258] This example, as illustrated in Figure 12, depicts exemplary images of a linear amplification process of the fluorescence signal intensity using benzophenone modified oligonucleotides of a single gene in a cell culture sample. The scale bar is 20 microns.

[0259] Approximately 100000 NIH3T3 cells were seeded onto a 24x60mm #1.5 glass coverslips. Cells were fixed with 4% PFA in lx PBS at room temperature for 10 min. After washing with lx PBS, samples were stored in 70% EtOH at -20C for permeabilization overnight. Samples w ere dried with nitrogen gas and a custom flow cell was attached to the glass coverslip. After rinsing with lx PBS, 7.5 mM BS-PEG5 in lx PBS was added for 30 min at room temperature. Samples were incubated with 100 mM N-( Propionyl oxy) succinimide in lx PBS two times for 30 min at room temperature. Samples were rinsed with 40% wash buffer (40% formamide, 2x SSC, 0.1% Triton-XlOO) three times. Primary probes were hybridized with a 40% hybridization buffer (40% formamide, 0.1 mg / mL yeast tRNA, 2X SSC, 10% 500kDa Dextran Sulfate) containing 5 nM / oligo at 37 C for 12 hours. After primary probe hybridization, the sample was rinsed with a 40% wash buffer 3 times andincubated at 37 C once. Subsequently, the sample was rinsed with 2X SSC solution. Then, 100 nM / oligo amplifier probes were hybridized to the sample in a 10% hybridization buffer (10% formamide, 2X SSC, 10% 6-10kDa Dextran Sulfate, 0.1% Triton-XlOO) at room temp for 30 min. The sample was rinsed with 10% wash buffer (Wash buffer: 10% formamide, 2X SSC, 0.1% TritonX-100) and lx PBS, then illuminated using a 365 nm UV lamp for 45 min. Next, samples were rinsed with 2x SSC and incubated with 1 uM of displacer strand in a 15% hybridization buffer at room temperature for 15 min. Post displacement, the sample was rinsed with a 20% wash buffer (20% formamide, 2X SSC, 0.1% TritonX-100). This process of amplifier hybridization, crosslinking, and displacement was repeated multiple times to obtain sufficient signal amplification. Samples ready to image were hybridized with a 10% hybridization buffer containing 100 nM / oligo readouts at room temperature for 15 min. Samples were stained with 3 ug / mL DAPI in 2X SSC for 1 min, then rinsed with 2X SSC twice. Anti-bleaching 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) was applied and samples were imaged using a 63x, 1.4 NA Leica objective and a spinning disk confocal microscope (Andor Dragonfly).

[0260] Figure 12 illustrates an exemplary embodiment of a fluorescent intensity signal increase over multiple rounds of linear amplification of benzophenone functionalized oligonucleotides.Example 13

[0261] Figure 13 illustrates an exemplary embodiment of a fluorescent intensity distribution change over multiple rounds of linear amplification of benzophenone functionalized oligonucleotides.Example 14

[0262] This example, as illustrated in Figure 14, depicts exemplary images of a linear and exponential amplification process of the fluorescence signal intensity using benzophenone modified oligonucleotides of a single gene in a cell culture sample.

[0263] Approximately 100000 NIH3T3 cells were seeded onto a 24x60mm #1.5 glass coverslips. Cells were fixed with 3% glyoxal, 0.8% acetic acid. 150 mM NaCl. and 45 mM NaOH at room temperature for 10 min. After washing with lx PBS, samples were stored in 70% EtOH at -20C for permeabilization overnight. Samples were dried with nitrogen gas and a custom flow cell was attached to the glass coverslip. After rinsing with lx PBS, the samplewas incubated with 0.1% NaBH4 for 15 minutes, and 7.5 mM BS-PEG5 in lx PBS was added for 15 min twice at room temperature. Samples were incubated with 100 mM N- (Propionyloxy) succinimide in lx PBS two times for 15 min at room temperature. Samples were rinsed with 50% wash buffer (50% formamide, 2x SSC, 0.1% Triton-XlOO) three times. Primary probes were hybridized with a 50% hybridization buffer (50% formamide, 0. 1 mg / mL yeast tRNA, 2X SSC, 10% 500kDa Dextran Sulfate) containing 5 nM / oligo at 37 C for 16 hours. After primary probe hybridization, the sample was rinsed with a 55% wash buffer 3 times and incubated at 37 C once. Subsequently, the sample was rinsed with 4x SSC solution. Then, 50 nM / oligo benzophenone-modified amplifier probes with 15 nt extender strands (reverse complement sequence between toehold and readout sites to make the amplifier probe partially double-stranded) were hybridized to the sample in a 10% hybridization buffer (10% formamide, 4X SSC, 10%, 40 kDa Dextran Sulfate, 0. 1% Triton- XlOO) for 45 min at 37 C. The sample was rinsed with 15% wash buffer (Wash buffer: 15% formamide, 2X SSC, 0.1% TritonX-100) and lx PBS, then illuminated using a 365 nm UV lamp for 5 min. Next, samples were rinsed with 55% wash buffer (55% formamide, 2x SSC, 0. 1% Triton-XlOO), then rinsed with 4x SSC and incubated with 1 uM of displacer strand in a 10% hybridization buffer at room temperature for 15 min. Post displacement, the sample was rinsed with a 25% wash buffer (25% formamide, 2X SSC, 0.1% TritonX-100). This process of amplifier hybridization, crosslinking, and displacement was repeated 8 times in this example experiment to obtain sufficient signal amplification in the linear amplification steps. The linear amplifier probes were targeted in the exponential amplification rounds. For exponential amplification rounds, 50-100 nM / oligo benzophenone-modified amplifier probes with 15 nt extenders were hybridized to the sample in a 10% hybridization buffer (10% formamide, 2X SSC. 10% 6-10kDa Dextran Sulfate, 0.1% Triton-XlOO) for 45 min. The sample was rinsed with 15% wash buffer (Wash buffer: 15% formamide, 2X SSC, 0. 1% TritonX-100) and lx PBS, then illuminated using a 365 nm UV lamp for 5 min. The sample was rinsed with a 55% wash buffer (55% formamide, 2X SSC, 0.1% TritonX-100), and the next round of benzophenone-modified amplifier probes were hybridized to the sample. The process was repeated 18 times in this example experiment with various imaging steps in between. Samples ready to image were hybridized with a 10% hybridization buffer containing 100 nM / oligo readout probes at room temperature for 15 min. Samples were stained with 3 ug / mL DAPI in 2X SSC for 1 min, then rinsed with 4X SSC twice. Antibleaching buffer (100 mM Tris-HCl pH 8. 4x SSC, 2 mM Trolox, 10% (w / v) D-Glucose, 1 : 100 Glucose Oxidase (200 U / rnL), 1 : 1000 Catalase) was applied and samples were imagedusing a 63x, 1.4 NA Leica objective and a spinning disk confocal microscope (Andor Dragonfly). Figure 14A illustrates an exemplary embodiment of a fluorescent intensity signal increase. Labelling above exemplary images shows number of total amplification rounds. Example image contrast is adjusted to illustrate the similarity of fluorescent signal features such as signal dot size and signal dot number in different amplification rounds. Each dot correspond to a signal from a single molecule of nucleic acid. Figure 14B illustrates an exemplary embodiment of a fluorescent intensity signal increase over multiple rounds of amplification of benzophenone functionalized oligonucleotides. In this exemplary image, peak intensities of individual dots were plotted for each imaging round, and the fold increase of fluorescence intensity is written above the respective data points.Example 15

[0264] Figure 15 illustrates additional designs for crosslinking amplification. Figure 15 A illustrates schematics of a sacrificial layer design for amplification. Primary probes were hybridized on a RNA molecule, followed by secondary and tertiary amplifier hybridization. In this scenario, the secondary amplifiers do not have crosslinking groups, but the tertiary amplifiers have crosslinking groups. After crosslinking the tertiary probes, a stringent stripping step was performed (60% formamide wash), which removed the secondary7amplifiers. Other probes that were not crosslinked were also be removed- such as the primary probes. The RNA could also be degraded without affecting the subsequent results.Additional steps of secondary and tertiary hybridization were performed, where the secondary amplifiers hybridize on the tertiary amplifiers that were crosslinked into the cell. The tertiary7amplifiers could hybridize to the secondary amplifiers. Stringent washing step was performed again to remove the secondary amplifiers. This left more tertiary amplifiers crosslinked near the target analyte. After n rounds of hybridizations, crosslinking and washes, tertiary^ amplifiers numbers increased near the target analyte. Figure 15B illustrates quantification of signal of individual amplified dot in cells implementing the scheme shown in Figure 15 A. After 3 rounds of secondary and tertiary hybridization, crosslinking and washing, a mean of 16.7-fold amplification was achieved. The images are shown for three amplifiers in (D) in NIH 3T3 cells using Eef2 as the target mRNA. The quantification was measured for amplifier 1. Each secondary7amplifier probe had two binding sites for the tertiary probe and each tertiary amplifier has two binding sites for the secondary amplifier probe. The histogram shows the intensities of the single dots in cells compared to single molecule FISH (smFISH) imaging without amplification. In this implementation, tertiaryamplifiers were crosslinked using Click reaction. Figure 15C illustrates a bridge adapter design that generates signal only when two amplified balls are physically proximal to each other. The highlighted bridge probe with a thicken line shows the bridge adapter that bind across two amplified balls. The thicken line represents a readout probe binding site. The amplification was performed with or without a sacrificial amplifier layer. The primary probes are designed to hybridize on adjacent regions on the target analytes, the RNA illustrated. Each of the primary probes contained amplifier binding sites such that two amplified balls result from adjacent pairs of amplifiers. Figure 15D illustrates images of cells amplified using the scheme shown in Figure 20C. 24 pairs of primary probes were used to target Eef2 mRNA in mammalian cell culture (NIH 3T3). Each pair contained primary probes that were hybridized on adjacent regions on the mRNA that are spaced one nucleotide apart. Each primary probe contained 2 amplifier binding sites for a total of 4 sites. Three of the sites were amplified. Amplifiers 1 (secondary and tertiary) and 2 (secondary7and tertiary) were on one primary7probe, while amplifier 3 (secondary and tertiary ) were on the adjacent primary probe. Amplification was performed using the sacrificial secondary layers which are removed by washes while the tertiary amplifiers are crosslinked to the cell by Click reactions. Many dots colocalize between amplifiers 1-3 indicating accurate amplification and detection of single molecules of mRNAs in cells. The bridge adapters across amplified balls generated by amplifiers 2 and 3 detected most of the same dots that colocalize across the three amplifier channels. In addition, non-specifically amplified dots in each of the amplified channels (left three panels) are not observed in the bridge adapter channel (right panel). The lower right comers of each image show zoomed-in images shown in the white box. The arrows in the insets on the lower right show regions where nonspecific dots appear in the individual amplified channel, but not in the bridge channel. Nonspecific amplification occurred because amplifiers, either secondary or tertiary probes, can stick to the cell non-specifically rather than the real analyte targets. These nonspecific events can be further amplified to give signal comparable to the real signals. However, because the nonspecific events are random, they are unlikely to occur at the same location for two different amplifier sequences, such as amplifier 2 and amplifier 3. Using bridge adapters that require two or more amplifier balls to be present in close proximity serves as a co-incidence detector that rejects nonspecific binding and detect only target analytes where the two primary' probes and the amplified products occur in close physical proximity. This approach when applied to different analytes present at close proximities, such as RNA-DNA. RNA-protein, DNA-protein. protein-protein or othermolecules, enabled detection of molecular interactions using amplification and bridge readouts.Example 16

[0265] This example, as illustrated in Figure 1 , depicts exemplary images of a linear and exponential amplification process of the fluorescence signal intensity using benzophenone modified oligonucleotides of a single gene in a cell culture sample. Cells were prepared as described in example 14. Figure 16A illustrates an exemplary embodiment of a fluorescent intensity signal increase for a small part of a recorded image to visualize individual fluorescence intensity peaks. Each dot corresponds to a signal from a single molecule of nucleic acid. Labelling above exemplary images shows number of total amplification rounds. Contrast is matched for all example images, with a 1 Ox shorter exposure time for the rightmost two images to illustrate the increase in signal intensity. Figure 16B illustrates an exemplary embodiment of a fluorescent intensity signal increase over multiple rounds of amplification of benzophenone functionalized oligonucleotides. In this exemplary image, peak intensities of individual dots were plotted for each imaging round, and the fold increase of fluorescence intensity is written above the respective data points.

[0266] Examples 17-20 describe RNA signal stabilization and amplification via clickchemistry.Example 17

[0267] In this Example, in one embodiment, the cell samples were prepared and fixed, with amine groups in the cells modified by activated ester groups using, for example, Alkyne- PEG4-NHS esters. For cell functionalization, besides adding the functional group to the cells via esterification, this could also be accomplished via a thiol or mercaptan nucleophile to make corresponding thioesters. In another embodiment, alkyne groups were attached to the cell where 3'-azide modified DNA oligos can crosslink to the cell via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reactions, preserving the spatial information and amplifying the signal of the unstable RNA, as shown in Figure 17A. For the crosslinking of probes, besides the CuAAC reaction described above, in one embodiment crosslinking can also be achieved using other established click-chemistry methods, including but not limited to strain-promoted azide-alkyne cycloadditions (SPAACs); tetrazine-trans-cyclooctene ligations(TCO-Tz; or inverse-electron demand Diels-Alder reactions); thiol-ene reactions; thiol-yne reactions; oxime ligations; hydrazone ligations; Diels-Alder reactions; and inverse-electrondemand Diels-Alder reactions (lEDDAs). Additionally, probes can be crosslinked to the cell via hydrazide treatment with carbonyls to form, for example, stable hydrazones.

[0268] Three amplification schemes based on the click-crosslinking of DNA probes to the cell: Split design, Branch design, and Padlock design were developed. For all three schemes, we designed two sets of amplifiers, named secondary and tertiary amplifiers. The tertiary amplifier can only bind to the secondary amplifier, and vice versa. This design prevents the aggregation of amplifiers in the hybridization solution before reaching the amplification site and enables a highly manageable amplification fold.Example 18

[0269] To eliminate noise, split probes were designed as shown in Figure 18A. Each tertiary amplifier binding requires the presence of both secondary amplifiers in close proximity, and each secondary amplifier binding requires primary probe pair or both tertiary amplifiers to be present in close proximity. This “double-lock” system significantly reduced noise caused by random amplifier sticking in the cell and subsequent amplification, as it is highly unlikely for the amplifier pair to stick at the same spot in the cell. The 3' end of DNA oligos (primary probe and amplifiers) were modified with an azide by adding azide-modified dATP using Terminal Deoxynucleotidyl Transferase (TdT). In this example, the cell samples were prepared and fixed, with amine groups in the cells modified by activated ester groups using Alkyne-PEG4-NHS esters. After each round of amplifier hybridization, sample was washed with 20% formamide wash buffer (20% Formamide, 0.1% Triton X-100 in 4x SSC) before clicking to the cell to remove redundant amplifiers.

[0270] The efficiency of CuAAC crosslinking by the design illustrated in Figure 17B. Stripping off the first cycle of click-crosslinked secondary amplifiers was attempted with a 60% formamide wash buffer (60% Formamide, 0.1% Triton X-100 in 4x SSC). Noncrosslinked amplifiers were washed off at room temperature, as shown in Figure 17C. The signal intensity was quantified before and after stripping. For the "clicked" sample, there was no significant decrease in signal intensity, whereas the "no click" sample showed barely detectable signal, as illustrated in Figure 17C-17D.

[0271] As shown in Figure 18B, RNA signal can be preserved and amplified with this amplification scheme. Signal could reach 5000 counts with 500ms exposure at 10% laserpower from a standard confocal microscope (Andor Dragonfly) while maintaining good colocalization with the smFISH signal.Example 19

[0272] An efficient amplification scheme was established, which allows branching out short amplifiers rapidly. The RNA binding site on primary probe was designed to be 30nt, amplifier binding sites were designed as 13nt / 15nt at both ends of the primary probe. After the hybridization process, excess probes were removed using a 30% formamide wash buffer (30% Formamide, 0.1% Triton X-100 in 4x SSC). The primary probes were then anchored to the cells at the 3' end via CuAAC. Amplification of the signal was achieved with branch amplifiers, which contain a 13nt / 15nt probe binding site at the 5’ end and two 13nt / 15nt amplification sites at the 3' end. To minimize noise, a 10% formamide wash was applied following each round of amplifier hybridization. In this example, the cell samples were prepared and fixed, with amine groups in the cells modified by activated ester groups using Alkyne-PEG4-NHS esters. The amplifiers were subsequently crosslinked to the cells through the CuAAC reaction to establish the branching structure. Redundant amplifiers were stripped off using a 60% formamide wash buffer post-crosslinking. For rapid amplification with the short branch amplifiers, each cycle of amplifier hybridization can be reduced from 1 hour to 30 mins. The amplification scheme is illustrated in Figure 19A.

[0273] The amplification efficiency was tested with 24 Eef2 primary probes. Sample was processed before signal amplification with the Branched scheme. After 6 cycles of amplification, we achieved a ~ 18x signal boost for 13nt amplifier binding and ~38x signal boost for 15nt amplifier binding compared to smFISH (Figure 19D). The highly amplified dots also colocalize extremely well in both 13nt BS and 15nt BS designs, as shown in Figure 19B-C. Therefore, we demonstrated that the Branch scheme can give rapid and high precision amplification.Example 20

[0274] In this scheme, all probes were designed in a padlock configuration, allowing for exonuclease digestion to reduce noise. Primary probes were designed with a 21nt RNA binding site at each end of the padlock. After hybridization, excess probes were washed away with a 30% formamide wash buffer (30% Formamide, 0.1% Triton X-100 in 4x SSC) and digested with exonuclease. Subsequently, the primary probes were crosslinked to the cell via CuAAC.

[0275] The signal was then amplified using azide-modified padlocked amplifiers, with 15nt probe binding sites at each end and two 30nt amplification sites designed in the middle for exponential amplification. For noise reduction, a 10% formamide wash and exonuclease digestion were performed after each round of amplifier hybridization. These amplifiers were also crosslinked to the cells using the CuAAC reaction to build up the structure. Excess and non-crosslinked amplifiers were washed away with a 60% formamide wash buffer after the crosslinking. The amplification scheme is shown in Figure 20A.

[0276] Different sites on the same primary probe were tested as to whether they could be amplified simultaneously. A single probe targeting the Eef2 gene was designed, which has three amplifier binding sites. After 14 cycles of amplification, the signals in different channels colocalize remarkably well, as presented in Figure 20B. Moreover, we quantified the dot intensity and achieved an ~8x signal boost compared to standard smFISH which has 24 primary probes (Figure 20C). Primary probes are expected to have 80% binding efficiency, therefore the amplification fold for the single probe is estimated to be ~150x. The strong colocalization and significant amplification of signal from the single probe experiment enables the detection of short RNA and large-scale barcoded experiments.REFERENCES

[0277] Additional background information can be found in the following references, each of which is hereby incorporated by reference in its entirety.

[0278] 1 . Moses, L. & Pachter, L. Museum of spatial transcriptomics. Nat Methods 19,534-546 (2022).

[0279] 2. Rouhanifard. S. H. et al. ClampFISH detects individual nucleic acid molecules using click chemistry-based amplification. Nat Biotechnol 37, 84-89 (2019).

[0280] 3. Dardani, I. et al. ClampFISH 2.0 enables rapid, scalable amplified RNA detection in situ. Nat Methods 19, 1403-1410 (2022).

[0281] 4. Wu, C. et al. RollFISH achieves robust quantification of single-molecule RNA biomarkers in paraffin-embedded tumor tissue samples. Commun Biol 1, 1-8 (2018).

[0282] 5. Wang, X. et al. Three-dimensional intact-tissue sequencing of single-cell transcriptional states. Science 361, eaat5691 (2018).

[0283] 6. Zeng, H. et al. Integrative in situ mapping of single-cell transcriptional states and tissue histopathology' in a mouse model of Alzheimer’s disease. Nat Neurosci 26, 430- 446 (2023).

[0284] 7. Gyllborg, D. et al. Hybridization-based in situ sequencing (HyblSS) for spatially resolved transcriptomics in human and mouse brain tissue. Nucleic Acids Research 48, el 12 (2020).

[0285] 8. Lee, J. H. et al. Highly Multiplexed Subcellular RNA Sequencing in Situ.Science 343, 1360-1363 (2014).

[0286] 9. Shah, S., Lubeck, E., Zhou, W. & Cai, L. In Situ Transcription Profiling ofSingle Cells Reveals Spatial Organization of Cells in the Mouse Hippocampus. Neuron 92, 342-357 (2016).

[0287] 10. Choi, H. M. T., Beck, V. A. & Pierce, N. A. Next-Generation in SituHybridization Chain Reaction: Higher Gain, Lower Cost, Greater Durability. ACS Nano 8, 4284-4294 (2014).

[0288] 11. Xia, C., Babcock, H. P., Moffitt, J. R. & Zhuang, X. Multiplexed detection ofRNA using MERFISH and branched DNA amplification. Sci Rep 9, 7721 (2019).

[0289] 12. He. S. et al. High-plex Multiomic Analysis in FFPE at Subcellular Level bySpatial Molecular Imaging. (2022) doi. https: / / doi.org / 10.1101 / 2021. l l.03.467020

Claims

CLAIMSWhat is claim is:

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

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

3. The method of claim 1 , wherein signals are detected by imaging or sequencing.

4. The method of claim 1, wherein the method comprises at least one step of crosslinking one or more amplifier probes to a cellular component.

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

6. The method of claim 5, wherein the click chemistry is selected from: strain-promoted azide-alkyne cycloadditions (SPAACs), tetrazine-trans-cyclooctene ligations (TCO- Tz), thiol-ene reactions, thiol-yne reactions, oxime ligations, hydrazone ligations, and Diels-Alder reactions; and inverse-electron demand Diels-Alder reactions (lEDDAs).

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

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

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

10. The method of claim 1, wherein one or more amplifier probes comprises a secondary, tertiary, and quaternary amplifier probe.

11. The method of claim 10, wherein, the quaternary' amplifier probe is identical to the secondary’ amplifier probe.

12. The method of claim 10, wherein the quaternary' amplifier probe interacts with the secondary’ amplifier probe.

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

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

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

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

17. The method of claim 1, wherein the samples are imaged after contacting one or more amplifier probes with one or more readout probes.

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

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

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

21. The method of claim 1, wherein the target analytes are selected from transcripts, RNA, DNA loci, chromosomes, DNA, proteins, peptides, lipids, glycans, cellular component, small molecules, metabolites, primary probe, amplifier probe, organelles and any combinations 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 at 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 each other.

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

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

29. The method of claim 28, wherein the amplifier probe binding sites are the same as each other.

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

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

32. The method of claim 1, wherein the amplifier probe comprises a primary probe binding site, a moiety that allows unbinding of the amplifier probe from the primary' probe, one or more of the same amplifier probe binding sites, one or more different amplifier probe binding sites, one or more of the same readout probe binding site, one or more different readout probe binding site, one or more of different crosslinking 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 amplifier probes are crosslinked to the cellular component by one or more optically dependent chemical moieties.

34. The method of claim 33, wherein the optically dependent chemical moieties are selected from diazirines, benzophenones, aryl-azides, or any combination thereof.

35. The method of claim 33. wherein one or more amplifier probes are crosslinked to one or more cellular components by exposure to light at a set wavelength, a set intensity, a set duration, or any combination thereof.

36. The method of claim 1, wherein one or more amplifier probes are crosslinked to the cellular component by esters.

37. The method of claim 36, wherein the esters are NHS-esters.

38. The method of claim 1, wherein one or more amplifier probes are crosslinked to the cellular component by cysteine reactive reagents.

39. The method of claim 38, wherein the reactive agents are maleimides.

40. The method of claim 1 , wherein one or more amplifier probes are crosslinked to the cellular component by peptide coupling reagents.

41. The method of claim 40, wherein the peptide coupling reagents are selected from carbodiimides, uronium salts, phosphonium salts, or any combination thereof.

42. The method of claim 1 , wherein one or more amplifier probes are crosslinked to the cellular component by in situ carbene generation.

43. The method of claim 1, wherein one or more amplifier probes are crosslinked to the cellular component by contacting the analyte with one or more rounds of crosslinkers.

44. The method of claim 43, wherein the crosslinkers are selected from paraformaldehyde (PFA), PEGylated bis (sulfosuccinimidyl) suberate (BSPEG). or combinations thereof.

45. The method of claim 1, wherein one or more amplifier probes are crosslinked to the cellular component by crosslinking cysteines by thiol modified oligonucleotides.

46. The method of claim 45, further comprising reacting the amplifier probe with BM(PEG)n.

47. The method of claim 1, wherein one or more amplifier probes are crosslinked to the cellular component by amine oligo or thiol oligo.

48. The method of claim 47, further comprising reacting the lysines, cysteines, or combinations thereof with using SM(PEG)n.

49. The method of claim 1 , wherein one or more amplifier probes are cross-linked to the cellular component, the components 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 components are primed to react with reactive moieties on the one or more amplifier probes.

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

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

53. The method of claim 1, wherein one or more amplifier probes are crosslinked to the cellular component by a Heck coupling, via A-h al os uccin imides and alkene reactions using a palladium catalyst.

54. The method of claim 53, wherein one or more amplifier probes are crosslinked to the cellular component by using small molecule substrates coupled to a readout probe.

55. The method of claim 1, wherein one or more amplifier probes are crosslinked to the cellular component with a functionalized hydrogel comprising a functional group which can react with a modified amplifier probe.

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

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

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

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

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

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

62. The method of claim 1, wherein one or more amplifier probes comprise 5' or 3' cross linkable molecules.

63. The method of claim 62, wherein the cross-linkable molecules are photo-cross- linkable molecules.

64. The method of claim 1 , wherein one or more amplifier probes comprise reverse complementary binding sites to one or more primary probe binding sites.

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

66. The method of claim 1 , wherein one or more amplifier probes are separated by washing, displacement, cleavage, photocleavage, chemical reduction, chemical degradation, enzymatic digestion, enzymatic reactions modifying the amplifier, or any combination thereof.

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

68. The method of claim 67, wherein one or more displacement probes compete with an interaction between the primary7probe and the amplifier probe.

69. The method of claim 1, wherein one or more amplifier 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 amplifier probes are separated by binding one or more displacement probes, the one or more displacement probes crosslinking to the cellular component.

71. The method of claim 1, further comprising detecting signals generated from an interaction of two amplicons.

72. The method of claim 71, further comprising contacting the sample with an adapter or bridge probe to generate a signal, the signal indicating the physical proximity7of tw o or more analytes.

73. The method of claim 71. further comprising contacting the sample with an adaptor or bridge probe to generate a signal, the signal indicating the physical proximify of tw o or more primary7probes.

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

75. The method of claim 74, wherein the products generate a signal that functions as a coincidence detector.

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

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

78. The method of claim 1, wherein one or more amplifier probes are separated by lightbased cleavage of the amplifier probe.

79. The method of claim 70, further comprising washing steps, wherein the cleaved amplifier probes are separated from the analyte.

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

81. The method of claim 1 , wherein one or more amplifier probes are separated by reducing a disulfide bridge within a secondary, or a tertiary probe interacting with an amplifier probe to cleave it.

82. The method of claim 1 , wherein one or more amplifier probes are separated by cleaving a pH-dependent moiety within a secondary, or a tertiary probe interacting with the amplifier probe.

83. The method of claim 1, wherein one or more amplifier probes are separated by sitespecific protease cleavages of the amplifier probe by TEV Protease, ribonuclease, TALEN, Zinc Finger protein, lipase, or endoglycosidase.

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

85. The method of claim 1, wherein one or more amplifier probes are separated by enzymatic cleavage of the amplifier probe at recognition sites, or restriction enzymes cleaving double-stranded probes.

86. The method of claim 85, wherein a Uracil-Specific Excision Reagent (USER) enzyme cleaves the amplifier probe.

87. The method of claim 1, wherein CRISPR Cas9 modification separates the amplifier probe.

88. The method of claim 1, wherein one or more amplifier probes are separated by targetspecific endonuclease to cleavage of the amplifier probes.

89. The method of claim 1, wherein one or more amplifier probes are separated by partial or complete removal of a secondary, or a tertian- probe interacting with the amplifier probe.

90. The method of claim 1 , wherein one or more amplifier 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 buffered or aqueous solution.

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