Compositions and methods for spatial analysis of multicell suspensions

Abstract

Described herein are compositions and methods for the evaluation of interactions between two or more cells within a cell suspension by spatial transcriptomics. The methods as disclosed herein may be useful for detecting or analyzing cellular interactions in a sample with two or more cells in a cell suspension. The methods as disclosed herein may comprise adhering cells to a surface in a cell suspension and further analysis for the detection of an analyte in the cell suspension.

Description

COMPOSITIONS AND METHODS FOR SPATIAL ANALYSIS OF MULTICELL SUSPENSIONSBACKGROUND

[0001] Described herein are compositions and methods related to analyzing cell suspensions (e.g., two or more cells) interactions while retaining spatial information for a detected analyte of interest. There is a need for understanding the interactions between two or more cells in their native context (e.g., in a native tissue).SUMMARY

[0002] In various aspects the present disclosure provides a method for measuring an interaction between two or more cells in a cell suspension comprising: (i) depositing the two or more cells onto a surface forming the cell suspension on the surface, (ii) performing a spatial analysis of the two or more cells, and (iii) analyzing the spatial analysis to determine the interaction between the two or more cells.

[0003] In some aspects, the surface is a planar surface. In some aspects, the surface is planar glass. In some aspects, the surface is coated with a coating. In some aspects, the coating comprises a biocompatible coating. In some aspects, the biocompatible coating is poly-L-lysine (PLL), aminosilane, hydrogel, epoxysilane, or any combination thereof. In some aspects, wherein the spatial analysis comprises spatial transcriptomics.

[0004] In some aspects, the spatial transcriptomics comprises a multiplex method for detecting different analytes in a sample by sequential signal-encoding of said analytes, comprising the steps of: (A) contacting the sample with at least twenty (20) different sets of analyte-specific probes for encoding of at least 20 different analytes, each set of analyte-specific probes interacting with a different analyte, wherein if the analyte is a nucleic acid each set of analyte-specific probes comprises at least five (5) analyte-specific probes which specifically interact with different substructures of the same analyte, each analyte-specific probe comprising: (aa) a binding element (S) that specifically interacts with one of the different analytes to be encoded, and (bb) an identifier element (T) comprising a nucleotide sequence which is unique to the analyte to be encoded (unique identifier sequence), wherein the analyte-specific probes of a particular set of analytespecific probes differ from the analyte-specific probes of another set of analyte-specific probes in the nucleotide sequence of the identifier element (T), wherein the analyte-specific probes in each set of analyte-specific probes binds to the same analyte and comprises the same nucleotide sequence of the identifier element (T) which is unique to said analyte; and (B) contacting the sample with at least one set of decoding oligonucleotides per analyte, wherein in each set ofdecoding oligonucleotides for an individual analyte each decoding oligonucleotide comprises: (aa) an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set, and (bb) a translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide; wherein the decoding oligonucleotides of a set for an individual analyte differ from the decoding oligonucleotides of another set for a different analyte in the first connect element (t); and (C) contacting the sample with at least a set of signal oligonucleotides, each signal oligonucleotide comprising: (aa) a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of a translator element (c) comprised in a decoding oligonucleotide, and (bb) a signal element, (D) detecting the signal caused by the signal element; (E) selectively removing the decoding oligonucleotides and signal oligonucleotides from the sample, thereby essentially maintaining the specific binding of the analyte-specific probes to the analytes to be encoded; (F) performing at least three (3) further cycles comprising steps B) to E) to generate an encoding scheme with a code word per analyte, wherein in particular the last cycle may stop with step (D).

[0005] In some aspects, the analyte comprises a nucleic acid. In some aspects, the nucleic acid is an mRNA molecule. In some aspects, the two or more cells comprise cells dissociated from tissue. In some aspects, the two or more cells comprise non-adherent cells. In some aspects, the two or more cells comprise adherent cells. In some aspects, the two or more cells comprise a rare cell type or a population of cells comprising less than 20 of a cell type. In some aspects, the two or more cells comprise cells that have been labeled with one or more dyes. In some aspects, the two or more cells comprise live cells. In some aspects, wherein the two or more cells comprise dead cells.

[0006] In some aspects, the two or more cells comprise cells that interact with at least one cell in the cell suspension. In some aspects, the two or more cells comprise an additional reagent that induces interactions between cells in the cell suspension. In some aspects, the two or more cells comprise an antibody. In some aspects, the two or more cells comprise tumor cells and immune cells. In some aspects, the two or more cells comprise a pharmaceutical molecule. In some aspects, the two or more cells comprise an engineered or transfected cell.INCORPORATION BY REFERENCE

[0007] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The novel features of the invention are set forth with particularity in the appended claims.

[0009] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0010] A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0011] FIG. 1 illustrates a schematic of a protocol for spatial transcriptomic analysis of individual cells in a cell suspension. The protocol includes sample preparation, slide preparation, cell attachment, optional storage, analysis by a spatial transcriptomic protocol, data analysis pipeline, and data collection.

[0012] FIG. 2 shows microscopic images captured of PBMC cells stained by violet and CFSE cell stains, the merged cell stain colors, and transcript detection from a spatial transcriptomic assay.

[0013] FIG. 3 shows a bar graph of the transcript count per tile for differentially treated samples of PBMC cell populations performed with different cell fixation times. Samples were pre-treated by i) no pre-treatment “direct”, ii) dried at room temperature and then frozen “dry freeze”, or iii) stored in ethanol at 4°C.

[0014] FIG. 4 shows a violin plot of the distribution of gene count and expression per cell and the cell size distribution.

[0015] FIG. 5 shows transcript detection of several genes, e.g., CD3E, NCAM1, ITGAX, FCER1 A, and MS4A1 in single cells in a cell suspension.

[0016] FIG. 6 shows a Uniform Manifold Approximation and Projection (uMAP) plot of different cell types identified in a PBMC cell suspension based on the detection of cell type specific gene expression by a spatial transcriptomics assay.

[0017] FIG. 7A and FIG. 7B show the uMAP plot of FIG. 6 overlayed with gene expression including CD3G, CCR7, CD40LG, CD8A, FOXP3, NCAM1, TRGC1, IL7R, GNLY, PRF1, CD14, FCGR3A, CD1C, LILRA4, TNFRSF17, and MS4A1.

[0018] FIG. 8 shows a scatter plot comparing differential gene expression between cell-to-cell interactions measured in a cell suspension.DETAILED DESCRIPTION

[0019] Described herein are methods for analyzing cell to cell interactions by detecting analytes with positional information. Previous techniques used for analyzing cell suspension interactions(e.g., interaction between two or more cells) remove the cells from their native tissue context, resulting in the loss of spatial information which is important for understanding the microenvironment and interactions in a cell suspension. Additionally, the process of removing cells from their native context may result in damaged cells, an impact in their gene expression profiles. Methods such as fluorescent-activated cell sorting (FACS) can be used to analyze cell suspensions, however, such methods also require separation of the cells from their original context and can result in cell damage (e.g., damage due to cell-shearing) and loss of rare or small population cell types. Additionally, previous assays (e.g., FACS) suffer from high technical variability introduced during the cell disassociation, sorting, and capture process needed for removing individual cells for analysis from a cell suspension. Methods to measure individual single cells from a cellular suspension commonly lack the resolution needed to accurately map transcripts back to their original place within the tissue thereby hampering the understanding of how the cellular gene expression may be influenced by local cell interactions in a suspension or in a tissue architecture. These limitations from previous cell suspension analysis methods demonstrate a need for methods to evaluate cell to cell interaction in a cell suspension or tissue by spatial biology methods (e.g., spatial genomics or spatial transcriptomics) to study the spatial relationships and heterogeneity within a suspension or tissue.

[0020] Described herein are compositions and methods that achieve analysis of cell-to-cell interactions within a cell suspension or tissue that preserve spatial information of transcriptomic data allowing for the evaluation of gene function and interaction within a native spatial context. The various embodiments as described herein provide insights into important cellular processes and gene regulatory networks capable of indicating potential therapeutic targets. Additionally, since the cells are not removed from their native context, high reproducibility and low variability can be achieved and rare or small population cell types can be studied which may play critical roles in health and disease.

[0021] As described herein, analysis of cell suspensions by spatial transcriptomics may be performed on a cell population immobilized on a surface (e.g., a glass slide). The spatial transcriptomics assay as described herein offers specificity and sensitivity in signal detection. The spatial transcriptomics assay as described herein is not limited by a minimum number of cells needed to perform an assay and may be performed on two or more cells. The spatial transcriptomics assay as described herein allows for the detection of cell-to-cell interactions and analysis of transcriptional effects of cellular interactions. In some embodiments, spatial transcriptomics may allow for the evaluation of cellular assays wherein a cell population is exposed to an effector and the gene expression is measured for the cells exposed to the effector,the cells in proximity to the cells exposed to the effector, the cells not in proximity to the effector, and any combination thereof.Cell Interactions

[0022] Compositions and methods of the present disclosure may be used for detecting or analyzing cellular interactions in a sample with two or more cells. Cellular interactions may be analyzed in cell suspensions or tissue samples. The measurement of cell-to-cell interactions may aid in understanding mechanisms of cell signaling, tissue development, immune responses, or cancer progression. These interactions are crucial for processes such as communication between immune cells during an immune response, coordination of cells during tissue regeneration, or the behavior of cancer cells in tumor microenvironments. Studying these interactions can provide insights into disease mechanisms, inform therapeutic strategies, or guide tissue engineering and regenerative medicine efforts.

[0023] In some embodiments, measuring cell-to-cell interactions is particularly important in the context of immune responses and cancer applications. For immune responses, understanding how immune cells like T cells, B cells, and antigen-presenting cells interact can reveal how the body defends itself against pathogens and how it may develop autoimmune conditions. In cancer research, analyzing interactions between cancer cells and the surrounding stromal cells, immune cells, or extracellular matrix can shed light on tumor growth, metastasis, and immune evasion. This knowledge is critical for developing targeted immunotherapies and improving cancer treatment outcomes.

[0024] In some embodiments, cell-to-cell interactions among various cell types may be measured to gain insights into a wide range of biological processes and disease mechanisms. In some embodiments, cells may be isolated from biological tissue (e.g., neuronal tissue, liver tissue, brain tissue, pancreas tissue, or a combination thereof). These cell types may include neuronal cells, liver cells, pancreas cells, peripheral blood mononuclear cells (PBMCs), such as lymphocytes (e.g., T cells, B cells, and NK cells), monocytes, dendritic cells, macrophages, or a combination thereof. Additionally, interactions among epithelial cells, endothelial cells, fibroblasts, and cancer cells may be analyzed. In some embodiments, cells are isolated from a tissue of interest. By studying how these diverse cells interact with each other, an understanding of processes such as antigen recognition, cytokine signaling, immune regulation, tissue regeneration, and tumor progression may be achieved. This approach may be useful in studying autoimmune diseases, infectious diseases, cancer, tissue engineering, and the effects of various therapies, including immunotherapies and regenerative medicine.

[0025] In some embodiments, spatial transcriptomics assays may be utilized to measure cell-to- cell interactions within a cell suspension by first preparing the cell suspension in an appropriate buffer or medium. For example, the cells may be resuspended in phosphate-buffered saline (PBS), a cell culture medium, or another suitable solution at a concentration that allows for adequate spacing between cells while still enabling interaction analysis. The cells may then be distributed onto a surface that allows for spatially resolved capture of analyte detection (e.g., gene expression through transcript detection). In some embodiments, the distribution of cells is a monolayer of cells. In some embodiments, the cells are distributed on a surface by smearing, pipetting and smearing, pipetting and spinning the surface, or by placing a droplet of cells onto a surface.

[0026] In some embodiments, the spatial arrangement of the cells within the suspension may be captured by assigning unique codewords to different analytes (e.g., transcripts) of the substrate by the methods as described herein. For example, the methods described herein allow for the detection of analyte (e.g., a transcript) based on a measured signal wherein each spot detected (e.g., signal detected) on the array corresponds to a specific spatial location, allowing the precise mapping of analyte detection (e.g., gene expression by transcript detection) to the location of individual cell within a cell suspensions (e.g., two or more cells).

[0027] In one example, immune cells be prepared in a suspension and analyzed using spatial transcriptomics to determine how their proximity affects gene expression related to immune activation, cytokine production, or cell signaling pathways. The spatial data obtained from these assays can reveal patterns of interaction that contribute to the coordination of the immune response.

[0028] In another example, cancer cells may be mixed with immune cells in a suspension to study how these interactions influence gene expression profiles related to immune evasion, metastasis, or drug resistance. For instance, the spatial transcriptomics assay may show how close proximity between cancer cells and macrophages leads to the upregulation of genes associated with tumor- associated macrophage (TAM) phenotypes, which are known to support tumor growth and suppress immune responses.

[0029] In some embodiments, the spatial transcriptomics data may be analyzed using computational tools that integrate the spatial coordinates with gene expression data to construct interaction networks, identify key signaling hubs, and predict functional outcomes based on cell- to-cell interactions within the suspension. This detailed analysis provides a powerful tool for understanding the complexities of cellular communication in environments where cells are free to interact without the constraints of tissue architecture.Cell Suspension Assay Methods

[0030] Methods as described herein allow for cell suspension analysis by sample preparation, cell fixation, spatial analysis (e.g., spatial transcriptomics), and data analysis. In some embodiments, sample preparation may comprise tissue culture, frozen cells, cells isolated from liquid biopsies, primary cells disassociated from tissues, or any combination thereof. In some embodiments, cells may be permeabilized prior to analysis. In some embodiments, the cells may be permeabilized in a suitable solvent (e.g., methanol, acetone, or a combination thereof). In some embodiments, the cells for analysis may be adhered to a surface (e.g., glass slides, polystyrene, glass coverslips, hydrogels, extracellular matrix-coated surfaces, or silicon or gold-coated surfaces). In some embodiments, cells may be adhered to glass slides. In some embodiments, cells may be adhered to a coated surface comprising a biocompatible coating (e.g., poly-L-lysine (PLL), aminosilane, hydrogel, epoxysilane, or any combination thereof). In some embodiments, cells may be adhered to a surface coated with materials such as collagen, fibronectin, laminin, poly-D-lysine, or Matrigel. Cells may be adhered to a surface by common cell fixation techniques (e.g., formaldehyde fixation, formamide fixation, alcohol fixation, acetone fixation, glutaraldehyde fixation, or any combination thereof). Cell adhered to a surface may be stored for further analysis (e.g., stored at 4°C or below). In some embodiments, cells are stored at 4°C, -20°C, -40°C, or - 80°C prior to analysis.

[0031] The methods of the present disclosure may involve attaching non-adherent cells to perform a functional interaction assay (e.g., a cell suspension assay). In some embodiments, this may include the immobilization and fixation of cells on a suitable substrate, such as multi-well slides.

[0032] In some embodiments, various buffers and solutions may be prepared for the process. For example, a buffer solution (e.g., PBS, HEPES, Tris), a fixation solution (e.g., formaldehyde, glutaraldehyde, methanol), and a storage solution (e.g., ethanol, methanol, isopropanol) may be prepared by combining the appropriate components in suitable proportions. The concentrations and volumes of these solutions may vary depending on the specific application.

[0033] Non-adherent cells may be resuspended in a buffer solution at an appropriate concentration (e.g., 1 x 106to 5 x 106cells / mL). If necessary, cells may be washed to remove any unwanted medium components (e.g., DMSO, serum). The cell suspension may be added to each well of the substrate, and the cells may be distributed evenly using methods such as gentle shaking or pipetting. The cells may then be immobilized by centrifugation (e.g., 200 x g to 400 x g for 3 to 10 minutes) or other suitable methods, followed by aspiration of the liquid.

[0034] Cells may be fixed by adding a fixation solution and incubating for a specified duration at a chosen temperature (e.g., 15 minutes to 2 hours at 4°C, room temperature, or 37°C). After fixation, the cells may be washed with a buffer solution and then treated with a storage solution(e.g., 70% ethanol, 90% methanol) at a specific temperature (e.g., 4°C, -20°C). The substrate with the treated cells may be stored or shipped under appropriate conditions.

[0035] For long-term storage, the substrate may be dried and stored at a low temperature (e.g., - 80°C). In some embodiments, the substrate may be stored in a preservation solution (e.g., ethanol, methanol) at a moderate temperature (e.g., 4°C, -20°C, or -80°C). The substrate may be transported by sealing it in an appropriate material (e.g., Parafilm, vacuum-sealed bag) and packaging it with cooling elements (e.g., ice packs, dry ice) to maintain the desired temperature (e.g., 4°C, -20°C, or -80°C) during transport.Methods of Measuring an Analyte in Cells within a Cell Suspension

[0036] Methods of the present disclosure include detecting and / or measuring an analyte in a sample (e.g., a cell suspension). In an embodiment of the disclosure the sample is a biological sample, preferably comprising biological tissue, further preferably comprising biological cells. A biological sample may be derived from an organ, organoids, cell cultures, stem cells, cell suspensions, primary cells, samples infected by viruses, bacteria, or fungi, eukaryotic or prokaryotic samples, smears, disease samples, a tissue section. Methods of the present disclosure include detecting an analyte by spatial *omics (e.g., spatial transcriptomics, spatial genomics, spatial proteomics, spatial metabolomics, etc.). In particular, the spatial transcriptomics detecting comprises a multiplex method for detecting different analytes in a sample by sequential signalencoding of said analytes as described in WO 2020 / 254519 Al, WO 2021 / 255244 and WO 2021 / 255263.

[0037] As mentioned above, spatial analysis according to the present disclosure means any kind of analysis where data from the cells are derived in a spatial manner. Spatial*omics may include the detection of small molecules compounds of tissues or cells, proteins, DNA, and / or RNA. More preferentially, spatial*omics is restricted to proteins, DNA, and / or RNA. More preferentially, spatial*omics is restricted to DNA and / or RNA. Even more preferentially, spatial*omics is restricted to smFISH. Even more preferentially, spatial*omics is restricted to any kind of sequential smFISH.

[0038] In some advantageous embodiments, the method according to the present invention is used for measuring an interaction between a plurality of cells (two or more cells) by spatial transcriptomics as the single molecule fluorescence in situ hybridization application. Typically, spatial transcriptomic analysis of biosafety samples is done after RNA or DNA is isolated. This is typical for a scenario using the Visium technology (lOx genomics) or the GeoMx system (Nanostring; Butler et al. (2021) “Shotgun transcriptome, spatial omics, and isothermal profilingof SARS-CoV-2 infection reveals unique host responses, viral diversification, and drug interactions” Nature Communications 12: 1660.

[0039] ). The analysis and detection of small quantities of analytes in biological and non- biological samples has become a routine practice in the clinical and analytical environment. Numerous analytical methods have been established for this purpose. Some of them use encoding techniques assigning a particular readable code to a specific first analyte which differs from a code assigned to a specific second analyte.

[0040] The method is particularly qualified to encode, identify, detect, count, or quantify analytes or single analytes molecules in a biological sample, such as a sample which contains nucleic acids as said analytes. It is understood that the biological sample may be in a form as it is in its natural environment (e.g., liquid, semi-liquid, solid, etc.), or processed environment, e.g., as a dried film on the surface of a device which may be re-liquefied before the method is carried out. Fixing the sample (e.g., biological tissue or biological cells) has the advantage that the analytes to be encoded, e.g., the nucleic acids, are immobilized and cannot escape. In doing so, the analytes then prepared for a better detection or encoding by the method according to the disclosure.

[0041] In yet a further embodiment within the set of analyte-specific probes the individual analyte-specific probes comprise binding elements (SI, S2, S3, S4, S5) which specifically interact with different sub-structures of one of the analytes to be encoded. By this measure the method becomes even more robust and reliable because the signal intensity obtained at the end of the method or a cycle, respectively, is increased. It is understood that the individual probes of a set while binding to the same analyte differ in their binding position or binding site at or on the analyte. The binding elements SI, S2, S3, S4, S5 etc. of the first, second, third fourth, fifth etc. analyte-specific probes therefore bind to or at a different position which, however, may or may not overlap.

[0042] In an embodiment, the present disclosure pertains to kit for multiplex analyte encoding, comprising:(A) at least twenty (20) different sets of analyte-specific probes for encoding of at least 20 different analytes, each set of analyte-specific probes interacting with a different analyte, wherein if the analyte is a nucleic acid each set of analyte-specific probes comprises at least five (5) analyte-specific probes which specifically interact with different sub-structures of the same analyte, each analyte-specific probe comprising:(aa) a binding element (S) that specifically interacts with one of the different analytes to be encoded, and(bb) an identifier element (T) comprising a nucleotide sequence which is unique to the analyte to be encoded (unique identifier sequence),wherein the analyte-specific probes of a particular set of analyte-specific probes differ from the analyte-specific probes of another set of analyte-specific probes in the nucleotide sequence of the identifier element (T), wherein the analyte-specific probes in each set of analyte-specific probes binds to the same analyte and comprises the same nucleotide sequence of the identifier element (T) which is unique to said analyte; and(B) at least one set of decoding oligonucleotides per analyte, wherein in each set of decoding oligonucleotides for an individual analyte each decoding oligonucleotide comprises:(aa) an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set, and(bb) a translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide; wherein the decoding oligonucleotides of a set for an individual analyte differ from the decoding oligonucleotides of another set for a different analyte in the identifier connect element (t); and(C) a set of signal oligonucleotides, each signal oligonucleotide comprising:(aa) a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of a translator element (c) comprised in a decoding oligonucleotide, and(bb) a signal element.

[0043] A multiplex method or assay allows for the simultaneously or sequential measurement of multiple analytes. According to the present disclosure it may be used to determine simultaneously or sequential the presence or absence of a plurality of predetermined (known) analytes like genomic as well as extra-genomic (e.g., mRNA) nucleic acid target sequences in a sample. An analyte may be “predetermined” in that its sequence is known to design a probe that binds to the that target.

[0044] In some embodiments according to the present disclosure at least 20, in particular at least 25, in particular at least 30 different analytes are detected and / or quantified in a sample in parallel. For example, there may be at least 5, at least 10, at least 20, at least 50, at least 75, at least 100, at least 300, at least 1,000, at least 3,000, at least 10,000, or at least 30,000 distinguishable analytespecific probes that are applied to a sample, e.g., simultaneously or sequentially. In some embodiments, there may be at least 100 analyte-specific probe sets (100 distinguishable analyte probes). In some embodiments, there may be at least 300 analyte-specific probe sets (100 distinguishable analyte probes).

[0045] In some embodiments for the multiplexing twenty (20) or more different sets of analytespecific probes for encoding of at least 20 different analytes or more are required, in particular more than 50, more than 100 or more than 200. In the multiplexing methods of the present disclosure, in particular at least 20 different groups of analytes (e.g., mRNA molecules).

[0046] In some embodiments, at least 4 rounds to collect information for identification of the analyte are carried out, wherein multiple readout increases the accuracy of identification and avoids false positives. The unique tag (e.g., a unique identifier sequence) can be identified by various techniques, including hybridization, e.g., with labeled probes, directly or indirectly or by sequencing (by synthesis, ligation). In particular, the identity of the tag can be encoded with one single signal (binary code), two or more signals, wherein the signal can be a fluorescent label (e.g., attached to an oligonucleotide).

[0047] In some embodiments, a kit does not comprise sets of analyte-specific probes as defined under item A).

[0048] Preferably, if the analyte in the kits or methods according to the present disclosure is a nucleic acid, each set of analyte-specific probes comprises at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40 different analyte-specific probes which specifically interact with different sub-structures of the same analyte. Nucleic acid analytes may include DNA molecules (e.g., genomic DNA, nuclear DNA, circular DNA, mitochondrial DNA, viral DNA, bacterial DNA, extra- or intracellular DNA) and / or RNA molecules (e.g., mRNA, hnRNA, miRNA, viral RNA, bacterial RNA, extra- or intracellular RNA, circular mRNA, tRNA, siRNA, snRNA, or rRNA).

[0049] In some embodiments according to the present disclosure the kit comprises at least two different sets of signal oligonucleotides, wherein a set of signal oligonucleotides comprises a different signal element and a different connector element (C) than a second set of signal oligonucleotides. For example, a first set of signal oligonucleotides may comprise signal oligonucleotides that comprise a different signal elements and a different connector element (C) than signal oligonucleotides comprised by a second set of signal oligonucleotides.

[0050] In particular, the kit may comprise at least two different sets of decoding oligonucleotides per analyte, wherein the decoding oligonucleotides comprised in these different sets comprise the same identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set, and wherein the decoding oligonucleotides of the different sets per analyte differ in the translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide.

[0051] In some embodiments the kit comprises at least two different sets of decoding oligonucleotides per analyte, wherein the decoding oligonucleotides comprised in these different sets comprise the same identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set, and wherein the decoding oligonucleotides of the different sets for at least one analyte differ in the translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide.

[0052] In some embodiments, in a given cycle, the number of different sets of decoding oligonucleotides (wherein each of the sets differ in at least the translator element (c)) per analyte corresponds to the number of different sets of signal oligonucleotides comprising different connector elements (C). However, the decoding oligonucleotides in a particular set of decoding oligonucleotides may interact with identical identifier elements (T) which are unique to a particular analyte. In some embodiments, sets of decoding oligonucleotides for the different analytes may comprise a common translator element(s) (c). For example, a first set of decoding oligonucleotides for a first analyte may have the same translator element (c) as a second set of decoding oligonucleotides for a second analyte.

[0053] In another aspect, the present disclosure is generally directed to methods including acts of exposing a sample to a plurality of analyte-specific probes; for each of the analyte-specific probes, determining binding of the analyte-specific probes within the sample; creating code words based on the binding of the analyte-specific probes, the decoding oligonucleotides and the signal oligonucleotides; and for at least some of the code words, matching the code word to a valid code word. In certain embodiments, this pattern of binding or hybridization of the analyte-specific probes, the decoding oligonucleotides and the signal oligonucleotides may be converted into a “code word.” For example, for instance, the code words may be “101” and “110” for a first analyte and a second analyte, respectively, where a value of 1 represents binding and a value of 0 represents no binding of decoding oligonucleotides and / or the binding of signal oligonucleotides without and / or quenched signal element. The analyte in the detection round / cycle is therefore not detectable during imaging.

[0054] To create such a zero (0) in a code word for an individual analyte the kit may comprise: (D) at least a set of non-signal decoding oligonucleotides for binding to a particular identifier element (T) of analyte-specific probes, wherein the decoding oligonucleotides in the same set of non-signal decoding oligonucleotides interacting with the same different identifier element (T), wherein each non-signal decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of aunique identifier sequence and does not comprise a translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide.

[0055] To create such a zero (0) in a code word for an individual analyte the kit may comprise: (D) at least a set of non-signal decoding oligonucleotides for binding to a particular identifier element (T) of analyte-specific probes, wherein the decoding oligonucleotides in the same set of non-signal decoding oligonucleotides interacting with the same different identifier element (T), wherein each non-signal decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of a unique identifier sequence and comprise a translator element that does not interact / bind to a signal oligonucleotide due to an instable binding sequence and / or due to the translator element is to short (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide.

[0056] In some embodiments, the kit comprises: (D) at least two (2) different sets of non-signal decoding oligonucleotides for binding to at least two different identifier elements (T) of analytespecific probes, each set of non-signal decoding oligonucleotides interacting with a different identifier element (T), wherein each non-signal decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of a unique identifier sequence and does not comprise a translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide.

[0057] In some embodiments, the different sets of non-signal decoding oligonucleotides may be comprised in a pre-mixture of different sets of non-signal decoding oligonucleotides or exist separately.

[0058] Furthermore, in some embodiments the kit may comprise: (E) a set of non-signal oligonucleotides, each non-signal oligonucleotide comprising: (aa) a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of the translator element (c), and (bb) a quencher (Q), a signal element and a quencher (Q), or does not comprise a signal element.

[0059] In some embodiments, the kit comprises: (E) at least two sets of non-signal oligonucleotides, each non-signal oligonucleotide comprising: (aa) a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of the translator element (c), and (bb) a quencher (Q), a signal element and a quencher (Q), or does not comprise a signal element.

[0060] In some embodiments, the different sets of non-signal oligonucleotides may be comprised in a pre-mixture of different sets of non-signal oligonucleotides or exist separately.

[0061] Further, in some embodiments the decoding oligonucleotides in a particular set of decoding oligonucleotides interacts with the same identifier elements (T) which are unique to a particular analyte.

[0062] In some embodiments, the different sets of decoding oligonucleotides may be comprised in a pre-mixture of different sets of decoding oligonucleotides or exist separately. In some embodiments, the different sets of analyte-specific probes may be comprised in a pre-mixture of different sets of analyte-specific probes or exist separately. In some embodiments, the different sets of signal oligonucleotides may be comprised in a pre-mixture of different sets of signal oligonucleotides or exist separately.

[0063] As mentioned above the analyte to be encoded may be a nucleic acid (e.g., DNA, PNA, RNA, or mixtures thereof). In some embodiments the analyte to be encoded may be RNA (e.g., mRNA, hnRNA, miRNA, viral RNA, bacterial RNA, extra- or intracellular RNA, circular mRNA, tRNA, siRNA, snRNA, rRNA, or any combination thereof). In some embodiments the analyte to be encoded may be mRNA.

[0064] The present disclosure pertains to a multiplex method for detecting different analytes in a sample by sequential signal-encoding of said analytes, comprising the steps of:(A) contacting the sample with at least twenty (20) different sets of analyte-specific probes for encoding of at least 20 different analytes, each set of analyte-specific probes interacting with a different analyte, wherein if the analyte is a nucleic acid each set of analyte-specific probes comprises at least five (5) analyte-specific probes which specifically interact with different substructures of the same analyte, each analyte-specific probe comprising:(aa) a binding element (S) that specifically interacts with one of the different analytes to be encoded, and(bb) an identifier element (T) comprising a nucleotide sequence which is unique to the analyte to be encoded (unique identifier sequence), wherein the analyte-specific probes of a particular set of analyte-specific probes differ from the analyte-specific probes of another set of analyte-specific probes in the nucleotide sequence of the identifier element (T), wherein the analyte-specific probes in each set of analyte-specific probes binds to the same analyte and comprises the same nucleotide sequence of the identifier element (T) which is unique to said analyte; and(B) contacting the sample with at least one set of decoding oligonucleotides per analyte, wherein in each set of decoding oligonucleotides for an individual analyte each decoding oligonucleotide comprises:(aa) an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set, and(bb) a translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide; wherein the decoding oligonucleotides of a set for an individual analyte differ from the decoding oligonucleotides of another set for a different analyte in the first connect element (t); and(C) contacting the sample with at least a set of signal oligonucleotides, each signal oligonucleotide comprising:(aa) a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of a translator element (c) comprised in a decoding oligonucleotide, and(bb) a signal element,(D) detecting the signal caused by the signal element;(E) selectively removing the decoding oligonucleotides and signal oligonucleotides from the sample, thereby essentially maintaining the specific binding of the analyte-specific probes to the analytes to be encoded;(F) performing at least three (3) further cycles comprising steps B) to E) to generate an encoding scheme with a code word per analyte, wherein in particular the last cycle may stop with step (D).

[0065] As mentioned above, the method according to the present disclosure comprises selectively removing the decoding oligonucleotides and signal oligonucleotides from the sample, thereby essentially maintaining the specific binding of the analyte-specific probes to the analyte to be encoded. All steps are performed sequentially. However, some steps may be performed simultaneously or sequential, the contacting steps A) to C), in particular B) and C).

[0066] By this measure the requirements for another round / cycle of binding further decoding oligonucleotides to the same analyte-specific probes are established, thus finally resulting in a code, or encoding scheme comprising more than one signal. This step is realized by applying conditions and factors well known to the skilled person, e.g., pH, temperature, salt conditions, oligonucleotide concentration, polymers etc.

[0067] In another embodiment of the present disclosure, the method may comprise repeating steps (B)-(E) at least three times to generate an encoding scheme. With this measure a code of four signals in case of four cycles / rounds which are carried out by the user, where 'n' is an integer representing the number of rounds. The encoding capacity of the method according to thedisclosure is herewith increased depending on the nature of the analyte and the needs of the operator. In an embodiment of the disclosure said encoding scheme is predetermined and allocated to the analyte to be encoded.

[0068] However, this measure enables a precise experimental set-up by providing the appropriate sequential order of the employed decoding and signal oligonucleotides and, therefore, allows the correct allocation of a specific analyte to a respective encoding scheme. The decoding oligonucleotides which are used in repeated steps (B)-(D2) may comprise a translator element (c2) which is identical with the translator element (cl) of the decoding oligonucleotides used in previous steps (B)-(E). In another embodiment of the disclosure decoding oligonucleotides are used in repeated steps (B)-(E) comprising a translator element (c2) which differs from the translator element (cl) of the decoding oligonucleotides used in previous steps (B)-(E). It is understood that the decoding elements may or may not be changed from round to round, i.e., in the second round (B)-(E) comprising the translator element c2, in the third round (B)-(E) comprising the translator element c3, in the fourth round (B)-(E) comprising the translator element c4 etc., wherein 'n' is an integer representing the number of rounds.

[0069] The signal oligonucleotides which are used in repeated steps (B)-(E) may comprise a signal element which is identical with the signal element of the decoding oligonucleotides used in previous steps (B)-(E). In a further embodiment of the disclosure signal oligonucleotides are used in repeated steps (B)-(E) comprising a signal element which differs from the signal element of the decoding oligonucleotides used in previous steps (B)-(E). In some embodiments no-signal oligonucleotides and / or no-signal decoding oligonucleotides for an individual analyte are used, resulting to the value 0 in the code word for this cycle / position. In some embodiments in a repeated cycle no decoding oligonucleotides for an individual analyte is contacted with the sample resulting also to the value 0 in the code word for this cycle / position.

[0070] By this measure each round the same or a different signal is provided resulting in an encoding scheme characterized by a signal sequence consisting of numerous different signals. This measure allows the creation of a unique code or code word which differs from all other code words of the encoding scheme. In another embodiment of the disclosure, the binding element (S) of the analyte-specific probe comprises a nucleic acid comprising a nucleotide sequence allowing a specific binding to the analyte to be encoded, preferably a specific hybridization to the analyte to be encoded.

[0071] In some embodiments, all steps are automated, in particular wherein steps B) to F) are automated, in particular by using a robotic system and / or an optical multiplexing system according to the present disclosure. In some examples, the steps may be performed in a fluidic system.

[0072] As mentioned above, with the methods according to the present disclosure an encoding scheme with a code word per analyte is generated. Therefore, each analyte may be associated with a specific code word, wherein said code word comprise a number of positions, and wherein each position corresponds to one cycle resulting in a plurality of distinguishable encoding schemes with the plurality of code words. In particular, said encoding scheme may be predetermined and allocated to the analyte to be encoded.

[0073] In some embodiments, the code words obtained for the individual analytes in the performed cycles comprise the detected signals and additionally at least one element corresponding to no detected signal like 0,1 or 0,1,2 etc.. No signal is detected for at least one analyte within at least one cycle if using a non-signal probe or a non-signal decoding oligonucleotide, or if in one cycle no decoding oligonucleotide is contacted with the corresponding identifier sequence comprised on analyte-specific probe interacting with the corresponding analyte in the sample. In this cycle the position has the value zero (0).

[0074] In some embodiments, at least for one individual analyte a position of the code word is zero (0). In particular, the code word zero (0) is generated by using no decoding oligonucleotides having an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of a corresponding analyte-specific probe for an individual analyte. As mentioned above, in some embodiments, if at least for one individual analyte a position of the code word is zero (0) in this cycle no corresponding decoding oligonucleotides having an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of a corresponding analyte-specific probe for an individual analyte are used.

[0075] Furthermore, in some embodiments the sample is contacted with at least two different sets of signal oligonucleotides, wherein the signal oligonucleotides in each set comprise a different signal element and comprise a different connector element (C).

[0076] In more embodiments, the sample is contacted with at least two different sets of decoding oligonucleotides per analyte, wherein the decoding oligonucleotides comprised in these different sets comprise the same identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set, and wherein the decoding oligonucleotides of the different sets per analyte differ in the translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide.

[0077] In more embodiments, the sample is contacted with at least two different sets of decoding oligonucleotides per analyte, wherein the decoding oligonucleotides comprised in these differentsets comprise the same identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set, and wherein the decoding oligonucleotides of the different sets per analyte differ in the translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide; wherein only one set of decoding oligonucleotides per analyte is used per cycle, and / or wherein different sets of decoding oligonucleotides are used in different cycles in combination with the corresponding set of signal oligonucleotides in the same cycle.

[0078] In some embodiments, the number of different sets of decoding oligonucleotides per analyte comprising different translator elements (c) corresponds to the number of different sets of signal oligonucleotides comprising different connector elements (C). All sets of decoding oligonucleotides for the different analytes may comprise the same type(s) of translator element(s) (c).

[0079] In some embodiments of the method according to the present disclosure, the sample is contacted with at least a set of non-signal decoding oligonucleotides for binding to a particular identifier element (T) of analyte-specific probes, wherein the decoding oligonucleotides in the same set of non-signal decoding oligonucleotides interacting with the same different identifier element (T), wherein each non-signal decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of a unique identifier sequence, and does not comprise a translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide.

[0080] As mentioned above, the sample may be contacted with at least two (2) different sets of non-signal decoding oligonucleotides for binding to at least two different identifier elements (T) of analyte-specific probes, each set of non-signal decoding oligonucleotides interacting with a different identifier element (T), wherein each non-signal decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of a unique identifier sequence, and does not comprise a translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide.

[0081] In some embodiments of the method according to the present disclosure, the different sets of non-signal decoding oligonucleotides may be comprised in a pre-mixture of different sets of non-signal decoding oligonucleotides or exist separately.

[0082] Furthermore, in some embodiments of the method according to the present disclosure, the sample is contacted with a set of non-signal oligonucleotides, each non-signal oligonucleotide comprising: (aa) a translator connector element (C) comprising a nucleotide sequence which isessentially complementary to at least a section of the nucleotide sequence of the translator element (c), and (bb) a quencher (Q), a signal element and a quencher (Q), or does not comprise a signal element.

[0083] In further embodiments, the sample may be contacted with: at least two sets of non-signal oligonucleotides, each non-signal oligonucleotide comprising: (aa) a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of the translator element (c), and (bb) a quencher (Q), a signal element and a quencher (Q), or does not comprise a signal element.

[0084] As mentioned above, the different sets of non-signal oligonucleotides may be comprised in a pre-mixture of different sets of non-signal oligonucleotides or exist separately.

[0085] In further embodiments, the decoding oligonucleotides in a particular set of decoding oligonucleotides interacts with identical identifier elements (T) which are unique to a particular analyte.

[0086] As mentioned above, the different sets of decoding oligonucleotides may be comprised in a pre-mixture of different sets of decoding oligonucleotides or exist separately as well as the different sets of analyte-specific probes may be comprised in a pre-mixture of different sets of analyte-specific probes or exist separately as well the different sets of signal oligonucleotides may be comprised in a pre-mixture of different sets of signal oligonucleotides or exist separately.

[0087] In some embodiments of the method according to the present disclosure, the binding element (S) comprise a nucleic acid comprising a nucleotide sequence allowing a specific binding to the analyte to be encoded, preferably a specific hybridization to the analyte to be encoded.

[0088] In some embodiments of the method according to the present disclosure, after step A) and before step B) the non-bound analyte-specific probes may be removed, in particular by washing, further after step B) and before step C) the non-bound decoding oligonucleotides may be removed, in particular by washing further, after step C) and before step D) the non-bound signal oligonucleotides may be removed, in particular by washing.

[0089] In some embodiments of the method according to the present disclosure, the analyte specific probes may be incubated with the sample, thereby allowing a specific binding of the analyte specific probes to the analytes to be encoded, further the decoding oligonucleotides may be incubated with the sample, thereby allowing a specific hybridization of the decoding oligonucleotides to identifier elements (T) of the respective analyte-specific probes, further the signal oligonucleotides may be incubated with the sample, thereby allowing a specific hybridization of the signal oligonucleotides to translator elements (T) of the respective decoding oligonucleotides.

[0090] As mentioned above, the analyte to be encoded may be a nucleic acid (e.g., DNA, PNA, RNA, or combinations thereof). In some embodiments, the analyte to be encoded may be a RNA (e.g., mRNA, hnRNA, miRNA, viral RNA, bacterial RNA, extra- or intracellular RNA, circular mRNA, tRNA, siRNA, snRNA, or rRNA).

[0091] By this measure the method is further developed to such an extent that the encoded analytes can be detected by any means which is adapted to visualize the signal element. Examples of detectable physical features include e.g., light, chemical reactions, molecular mass, radioactivity, etc.

[0092] In some embodiments, the signal caused by the signal element, therefore in particular the binding of the signal oligonucleotides to the decoding oligonucleotides, interacting with the corresponding analyte probes, bound to the respective analyte is determined by: imaging at least a portion of the sample; and / or using an optical imaging technique; and / or using a fluorescence imaging technique; and / or multi-color fluorescence imaging technique; and / or super-resolution fluorescence imaging technique.

[0093] Methods as disclosed herein may also comprise additional methods for detection of an additional analyte. In some embodiments, an additional analyte is detected that is a protein or peptide. In some embodiments, 10 protein targets, 20 protein targets, 30 protein targets, or 40 protein targets are detected in the sample in addition to another analyte (e.g., a nucleic acid). In some embodiments, various antibody staining techniques may be employed to detect and visualize specific proteins or antigens within a cell suspension. These techniques include direct and indirect staining, wherein antibodies either directly bind to antigens or use secondary antibodies for signal amplification. In some embodiments, fluorescent antibody staining is used. Multiplex staining allows for the simultaneous detection of multiple antigens. In some embodiments, the sample will be treated with a blocking agent (e.g., salmon sperm, BSA, goat serum) prior to analyte detection.

[0094] The kits and method according to the present disclosure may be used ideally for in vitro methods for diagnosis of a disease selected from the group comprising cancer, neuronal diseases, cardiovascular diseases, inflammatory diseases, autoimmune diseases, diseases due to a viral or bacterial infection, skin diseases, skeletal muscle diseases, dental diseases, and prenatal diseases.

[0095] Further, the kits and method according to the present disclosure may be used also ideally for in vitro methods for diagnosis of a disease in plants selected from the group comprising: diseases caused by biotic stress, preferably by infectious and / or parasitic origin, or diseases caused by abiotic stress, preferably caused by nutritional deficiencies and / or unfavorable environment.

[0096] Further, the kits and method according to the present disclosure may be used also ideally for in vitro methods for screening, identifying and / or testing a substance and / or drug comprising: (a) contacting a test sample comprising a sample with a substance and / or drug, (b) detectingdifferent analytes in a sample by sequential signal-encoding of said analytes with a method according to the present disclosure.

[0097] An optical multiplexing system suitable for the method according to the present disclosure, comprising at least: a reaction vessel for containing the kits or part of the kits according to the present disclosure; a detection unit comprising a microscope, in particular a fluorescence microscope; a camera; a liquid handling device.

[0098] In some embodiments, optical multiplexing system may comprise further a heat and cooling device and / or a robotic system.

[0099] In some embodiments, the method according to the present disclosure encodes a nucleic acid analyte, such as an mRNA, e.g., such an mRNA coding for a particular protein.

[0100] In some embodiments, the method described herein is used for specific detection of many different analytes in parallel. The technology allows to distinguish a higher number of analytes than different signals are available. The process includes at least four consecutive rounds of specific binding, signal detection and selective denaturation (if a next round is required), eventually producing a signal code. To decouple the dependency between the analyte specific binding and the oligonucleotides providing the detectable signal, a so called “decoding”- oligonucleotide is introduced. The decoding oligonucleotide transcribes the information of the analyte specific probe set to the signal oligonucleotides.

[0101] In a specific embodiment, the method may comprise the steps of (1) providing one or more analyte specific probe sets, the set of analyte specific probes consist of one or more different probes, each differing in the binding moiety that specifically interacts with the analyte, all probes of a single probe set are tethered to a sequence element (unique identifier), that is unique to a single probe set and allows the specific hybridization of a decoding oligonucleotide, (2) specific binding of the probe sets to their target binding sites of the analyte, (3) eliminating non-bound probes (e.g. by a wash step), (4) providing a mixture of decoding oligonucleotides that specifically hybridize to the unique identifier sequences of the probe sets, the decoding oligonucleotides comprise of at least two sequence elements, a first element that is complementary to the unique identifier sequences of the corresponding probe set and a second sequence element (translator element) that provides a sequence for the specific hybridization of a signal oligonucleotide, the translator element defines the type of signal that is recruited to the decoding oligonucleotide, (5) specific hybridization of the decoding oligonucleotides to the unique identifier sequences provided by the bound probe sets, (6) eliminating non-bound decoding oligonucleotides (e.g. by washing step), (7) providing a mixture of signal oligonucleotides, comprising of a signal that can be detected and a nucleic acid sequence that specifically hybridizes to the translator element of one of the decoding oligonucleotides used in the former hybridization step, (8) specifichybridization of the signal oligonucleotides, (9) eliminating non-bound signal oligonucleotides, (10) detection of the signals, (11) selective release of decoding oligonucleotides and signal oligonucleotides while the binding of specific probe sets to the analyte is almost or completely unaffected, (12) eliminating released decoding oligonucleotide and signal oligonucleotides (e.g., by a washing step) while the binding of specific probes sets to the analytes is almost or completely unaffected , repeating the steps 4 to 12 at least three times until the detection of a sufficient number of signals to generate an encoding scheme for each different analyte of interest.

[0102] It is to be understood that the before-mentioned features and those to be mentioned in the following cannot only be used in the combination indicated in the respective case, but also in other combinations or in an isolated manner without departing from the scope of the disclosure.

[0103] The method disclosed herein is used for specific detection of many different analytes in parallel. The technology allows distinguishing a higher number of analytes than different signals are available. The process preferably includes at least two consecutive rounds of specific binding, signal detection and selective denaturation (if a next round is required), eventually producing a signal code. To decouple the dependency between the analyte specific binding and the oligonucleotides providing the detectable signal, a so called “decoding” oligonucleotide is introduced. The decoding oligonucleotide transcribes the information of the analyte specific probe set to the signal oligonucleotides.

[0104] The present disclosure pertains further to methods of detecting an analyte, comprising: attaching a plurality of analyte-specific probes to the analyte, wherein the analyte-specific probes independently attach to the analyte and wherein the analyte-specific probes share a common identifier segment (T); annealing a plurality of first decoding oligonucleotides to the analytespecific probes, wherein the first decoding oligonucleotides share a first common region that is reverse complementary to the common identifier segment and a second common region; annealing a first signal oligonucleotide to at least one of the pluralities of first decoding oligonucleotides such that an oligo tethered to the first signal oligonucleotide is reverse complementary to the second common region; detecting the first signal oligonucleotide; removing the plurality of first decoding oligonucleotides; annealing a plurality of second decoding oligonucleotides to the analyte-specific probes, wherein the second decoding oligonucleotides share a first common region that is reverse complementary to the common identifier segment and a second decoding oligonucleotides second common region that differs from the second common region of the first decoding; annealing a second signal oligonucleotide to at least one of the pluralities of second decoding oligonucleotides such that an oligo tethered to the signal oligonucleotide is reverse complementary to the second decoding oligonucleotide second common region; and detecting the second signal oligonucleotide.

[0105] In the above-mentioned embodiment a second aliquot of a plurality of first decoding oligonucleotides is annealed to the analyte-specific probes. Furthermore, a first aliquot of a plurality of first decoding oligonucleotides is annealed to the analyte-specific probes.

[0106] In some embodiments, no second signal oligonucleotide to the at least one of the pluralities of first decoding oligonucleotides is annealed. No third signal oligonucleotide to the at least one of the pluralities of first decoding oligonucleotides is annealed.

[0107] In some further embodiments, the present disclosure pertains to a method of assigning an analyte to a position in an image, comprising assigning a fluorescence pattern to the analyte, observing the fluorescence pattern at the position in the image, and assigning the analyte to the position, in particular wherein observing the fluorescence pattern comprises repeating steps of labeling the position using a fluorophore tagged oligo drawn from a re-accessible pool, performing a single excitation at the position in the image, and contacting the analyte to a denaturant, in particular wherein observing the fluorescence pattern comprises repeating steps of labeling the position using a fluorophore tag-recruiting bridging oligo drawn from a re-accessible pool, performing a single excitation at the position in the image, and contacting the analyte to a denaturant.

[0108] In some further embodiments, the present disclosure pertains to a composition comprising a cell having nucleic acids distributed therein, wherein a first nucleic acid is tagged by a first plurality of probes that target adjacent segments of the first nucleic acid and that share a common first tether segment; a second nucleic acid is tagged by a second plurality of probes that target adjacent segments of the second nucleic acid and that share a common second tether segment; and a third nucleic acid is tagged by a third plurality of probes that target adjacent segments of the third nucleic acid and that share a common third tether segment; a first adapter population comprising molecules having a first tether reverse complementary region and a first fluorophore adapter tether; a second adapter population comprising molecules having a second tether reverse complementary region and a second fluorophore adapter tether; a third adapter population comprising molecules having a third tether reverse complementary region and a first fluorophore adapter tether; a population of first fluorophores having a first tether reverse complementary region; and a population of second fluorophores having a second tether reverse complementary region.

[0109] In some further embodiments, the present disclosure pertains to a method of assigning coded fluorescence patterns to a plurality of target analytes in a cell, comprising subjecting the cell to a plurality of detection rounds, each detection round comprising contacting the cell to representatives of the same at least two populations of tagged fluorescence moi eties, and removing the fluorescent moieties after a single excitation event, wherein the number of patterns detectableincreases exponentially with the number of detection rounds, wherein the fluorescence moieties are not tagged with nucleic acid tags that are specific to the target nucleic acids, and wherein separate aliquots of common tagged fluorescence moieties are used across multiple detection rounds.

[0110] With the above notified method a total decoding efficiency of at least 30% may be achieved.[OHl] In some further embodiments, the present disclosure pertains to a method of assigning coded fluorescence patterns to a plurality of target analytes in a cell, comprising: contacting a target to a bipartite labeling probe, the bipartite labeling probe comprising a target-specific moiety and a fluorophore-specifying moiety; contacting the bipartite labeling probe to a first aliquot of a fluorophore reservoir comprising no more than two populations of fluorophores; replacing the fluorophore specifying moiety in the bipartite probe, and contacting the bipartite labeling probe to a second aliquot of the fluorophore reservoir comprising the same no more than two populations.

[0112] In some embodiments of the above notified method, replacing the fluorophore specifying moiety in the bipartite probe comprises denaturing a binding between a target-specific moiety and a fluorophore-specifying moiety after subjecting the bipartite labeling probe bound to a fluorophore of the fluorophore to excitation energy. Replacing the fluorophore specifying moiety in the bipartite probe comprises drawing from one of no more than two fluorophore specifying moiety reservoirs.

[0113] In some further embodiments, the present disclosure pertains to a method of detecting an analyte, comprising: attaching a plurality of probes to the analyte, in particular a nucleic acid, wherein the probes independently attach / anneal to the analyte and wherein the probes share a common identifier segment; annealing a plurality of first adapter segments to the probes, wherein the first adapter segments share a first common region that is reverse complementary to the common identifier segment and a second common region, in particular configured to accommodate a single reporter / selected from no more than two reporter categories; annealing a first reporter to at least one of the plurality of first adapter segments such that an oligo tethered to the first reporter is reverse complementary to the second common region; detecting the first reporter; removing the plurality of first adapter segments, in particular without annealing a second reporter to the at least one of the pluralities of first adapter segments; annealing a plurality of second adapter segments to the probes, wherein the second adapter segments share a first common region that is reverse complementary to the common identifier segment and a second adapter second common region that differs from the second common region of the first adapter segments, in particular configured to accommodate a single reporter / selected from no more than tworeporter categories; annealing a second reporter to at least one of the pluralities of second adapter segments such that an oligo tethered to the second reporter is reverse complementary to the second adapter second common region; and detecting the second reporter, in particular without annealing a third reporter to the at least one of the pluralities of first adapter segments.

[0114] The analyte or target may be a nucleic acid, e.g., DNA or RNA, and the probe set comprises oligonucleotides that are partially or completely complementary to the whole sequence or a subsequence of the analyte (e.g., a nucleic acid sequence) to be detected. The nucleic acid sequence specific oligonucleotide probe sets comprising analyte-specific probes (1) including a binding element (S) that specifically hybridizes to the target nucleic acid sequence to be detected, and an identifier element (T) comprising a nucleotide sequence which is unique to said set of analyte-specific probes (unique identifier sequence).

[0115] In some embodiments, at least one analyte is a first nucleic acid and at least a second analyte is a second nucleic acid and at least the first probe set binds to the first nucleic acid sequence and at least the second probe set binds specifically to the second nucleic acid analyte. Other combinations are possible as well.

[0116] An embodiment of the general method of the present disclosure may be as follows. Step 1 : applying the at least 20 analyte- or target-specific probe sets. The target nucleic acid sequence is incubated with a probe set consisting of oligonucleotides with sequences complementary to the target nucleic acid. In this example, a probe set of 5 different probes is shown, each comprising a sequence element complementary to an individual subsequence of the target nucleic acid sequence (SI to S5). In this example, the regions do not overlap. Each of the oligonucleotides targeting the same nucleic acid sequence comprises the identifier element or unique identifier sequence (T), respectively. Step 2: Hybridization of the probe set. The probe set is hybridized to the target nucleic acid sequence under conditions allowing a specific hybridization. After the incubation, the probes are hybridized to their corresponding target sequences and provide the identifier element (T) for the next steps. Step 3 : Eliminating non-bound probes. After hybridization, the unbound oligonucleotides are eliminated, e.g., by washing steps. Step 4: Applying the decoding oligonucleotides. The decoding oligonucleotides consisting of at least two sequence elements (t) and (c) are applied. While sequence element (t) is complementary to the unique identifier sequence (T), the sequence element (c) provides a region for the subsequent hybridization of signal oligonucleotides (translator element). Step 5: Hybridization of decoding oligonucleotides. The decoding oligonucleotides are hybridized with the unique identifier sequences of the probes (T) via their complementary first sequence elements (t). After incubation, the decoding oligonucleotides provide the translator sequence element (c) for a subsequent hybridization step. Step 6: Eliminating the excess of decoding oligonucleotides. After hybridization, the unbounddecoding oligonucleotides are eliminated, e.g., by washing steps. Step 7: Applying the signal oligonucleotide. The signal oligonucleotides are applied. The signal oligonucleotides comprise at least one second connector element (C) that is essentially complementary to the translator sequence element (c) and at least one signal element that provides a detectable signal (F). Step 8: Hybridization of the signal oligonucleotides. The signal oligonucleotides are hybridized via the complementary sequence connector element (C) to the translator element (c) of decoding oligonucleotide. After incubation, the signal oligonucleotides are hybridized to their corresponding decoding oligonucleotides and provide a signal (F) that can be detected. Step 9: Eliminating the excess of signal oligonucleotides. After hybridization, the unbound signal oligonucleotides are eliminated, e.g., by washing steps. Step 10: Signal detection. The signals provided by the signal oligonucleotides are detected. The following steps (steps 11 and 12) may be unnecessary for the last detection round or may be necessary (e.g., if further measurements are performed on the sample). Step 11 : Selective denaturation. The hybridization between the unique identifier sequence (T) and the first sequence element (t) of the decoding oligonucleotides is dissolved. The destabilization can be achieved via different mechanisms well known to the trained person like for example: increased temperature, denaturing agents, etc. The target- or analytespecific probes are not affected by this step. Step 12: Eliminating the denatured decoding oligonucleotides. The denatured decoding oligonucleotides and signal oligonucleotides are eliminated (e.g., by washing steps) leaving the specific probe sets with free unique identifier sequences, reusable in a next round of hybridization and detection (steps 4 to 10). This detection cycle (steps 4 to 12) is repeated at least four times until the planed encoding scheme is completed.

[0117] The “analyte-specific probe” (e.g., “probe”) comprises at least two elements, the so-called binding element (S) which specifically interacts with (e.g., is essentially complementary to, binds to, hybridizes to, or a combination thereof) an analyte, and a so-called identifier element (T) comprising a “unique identifier sequence”. The binding element (S) may be a nucleic acid such as a hybridization sequence or an aptamer.

[0118] The “unique identifier sequence” as comprised by the analyte-specific probe is unique in its sequence compared to other unique identifiers. “Unique” in this context means that it specifically identifies only one analyte, e.g., a nucleic acid, or, alternatively, it specifically identifies only a group of analytes, e.g., a group of nucleic acids, independently whether the group of analytes comprises a gene family or not. Therefore, the analyte or a group of analytes to be encoded by this unique identifier can be distinguished from all other analytes or groups of analytes that are to be encoded based on the unique identifier sequence of the identifier element (T). Or, in other words, there is only one “unique identifier sequence” for a particular analyte or a group of analytes, but not more than one, e.g., not even two. Due to the uniqueness of the unique identifiersequence the identifier element (T) hybridizes to one type of decoding oligonucleotides during a cycle. The length of the unique identifier sequence is within the range 8-60 nt, 12-40 nt, or 14-20 nt, which may depend on the number of analytes encoded in parallel and the stability of interaction needed. A unique identifier may be a sequence element of the analyte-specific probe, attached directly or by a linker, a covalent bond or high affinity binding modes, e.g., antibody-antigen interaction, streptavidin-biotin interaction etc. It is understood that the term “analyte specific probe set” includes a plurality of analyte specific probes which may differ in their binding elements (S) in a way that each probe binds to the same analyte but possibly to different parts thereof, for instance to different (e.g., neighboring) or overlapping sections of the nucleotide sequence comprised by the nucleic acid molecule to be encoded. However, each of the plurality of the probes comprises the same identifier element (T). For example, an analyte specific probe set may comprise a plurality of analyte specific probes that each independently comprise a different binding element (S) and a common identifier element (T). The different binding elements (S) comprised by the plurality of analyte specific probes in an analyte specific probe set may bind different regions (e.g., different sequences) comprised by an analyte (e.g., a nucleic acid).

[0119] A “bipartite labeling probe” comprises a binding sequence capable of hybridizing the analyte and a binding probe sequence capable of binding a detectable signal molecule like a fluorophore or a nucleic acid sequence comprising a fluorophore.

[0120] A “decoding oligonucleotide” or an “adapter” or a / adapter segment” comprises at least two sequence elements, one sequence element that can specifically bind to a unique identifier sequence, referred to as an “identifier connector element” (t) or “first connector element” (t), and a second sequence element specifically binding to a signal oligonucleotide, referred to as “translator element” (c). The length of the sequence elements is within the range 8-60 nt, 12-40 nt, 14-20 nt, which may be dependent on the number of analytes to be encoded in parallel, the stability of interaction needed, the number of different signal oligonucleotides used, or a combination thereof. The length of the two sequence elements may or may not be the same.

[0121] A “signal oligonucleotide” or a “reporter” as used herein comprises at least two elements, a so-called “translator connector element” (C) or "second connector element" (C) having a nucleotide sequence specifically hybridizable to at least a section of the nucleotide sequence of the translator element (c) of the decoding oligonucleotide, and a “signal element” which provides a detectable signal. This element can either actively generate a detectable signal or provide such a signal via manipulation, e.g., fluorescent excitation. Typical signal elements are, for example, enzymes that catalyze a detectable reaction, fluorophores, radioactive elements, or dyes.

[0122] A “set” refers to a plurality of moi eties or subjects, e.g., analyte-specific probes or decoding oligonucleotides, whether the individual members of said plurality are identical ordifferent from each other. In an analyte specific probe set, the analyte specific probes are identical in the identifier element (T) but may comprise a different binding element (S) for specifically interacting with the same analyte but for specifically interacting with different sub-structures of the same analyte to be encoded.

[0123] An “analyte specific probe set” refers to a plurality of moi eties or subjects, e.g., analytespecific probes that are different from each other and bind to independent regions of the analyte. A single analyte specific probe set is further characterized by the same unique identifier.

[0124] A “decoding oligonucleotide set” refers to a plurality of decoding oligonucleotides specific for a certain unique identifier needed to realize the encoding independent of the length of the code word. Each and all of the decoding oligonucleotides included in a “decoding oligonucleotide set” bind to the same unique identifier element (T) of the analyte-specific probe.

[0125] In this disclosure “simultaneously detecting” means that all probes detecting the analyte may be added in one detection round. For example, genomic as well as mRNA may be detected in one detection round. In this disclosure “sequential detecting” means that the probes detecting the analyte may be added sequentially, e.g., probe set 1 for detecting analyte 1 is added first, then, optionally after a washing step, probe set 2 for detecting analyte 2 is added second, etc. For example, first, genomic nucleic acid elements may be detected in round 1, then followed by the detection of nucleic acid elements of mRNA in round 2, optionally, with a washing step in between round 1 and round 2. In some embodiments both “simultaneously detecting” as well as “sequential detecting” may be performed, e.g., detecting simultaneously analyte 1 and 2 with a first and second set of probes, the, optionally performing a washing step, and then detecting simultaneously analyte 3 and 4 with a third and fourth set of probes.

[0126] In certain embodiments, this pattern of binding or hybridization of the decoding oligonucleotides may be converted into a “code word.” For example, the code words could be also " 101 " and " 110" for an analyte, where a value of 1 represents binding and a value of 0 represents no binding. The code words may also have longer lengths in other embodiments. A code word can be directly related to a specific unique identifier sequence of an analyte-specific probe. Accordingly, different analyte-specific probe may match certain code words, which can then be used to identify the different analytes of the analyte-specific probe based on the binding patterns of the decoding oligonucleotide. However, if no binding is evident, then the code word would be "000" in this example.

[0127] The values in each code word can also be assigned in different fashions in some embodiments. For example, a value of 0 could represent binding while a value of 1 represents no binding. Similarly, a value of 1 could represent binding of a secondary nucleic acid probe with one type of signaling entity while a value of 0 could represent binding of a secondary nucleic acidprobe with another type of distinguishable signaling entity. These signaling entities could be distinguished, for example, via different colors of fluorescence. In some cases, values in code words need not be confined to 0 and 1. The values could also be drawn from larger alphabets, such as ternary (e.g., 0, 1, and 2) or quaternary (e.g., 0, 1, 2, and 3) systems. Each different value could, for example, be represented by a different distinguishable signaling entity, including (in some cases) one value that may be represented by the absence of signal.

[0128] The code words for each analyte may be assigned sequentially or may be assigned at random. For instance, a first analyte may be assigned to 101, while a second nucleic acid target may be assigned to 110. In addition, in some embodiments, the code words may be assigned using an error-detection system or an error- correcting system, such as a Hamming system, a Golay code, or an extended Hamming system (or a SECDED system, i.e., single error correction, double error detection). Such systems can be used to identify where errors have occurred, and in some cases, such systems can also be used to correct the errors and determine what the correct code word should have been. For example, a code word such as 001 may be detected as invalid and corrected using such a system to 101, e.g., if 001 is not previously assigned to a different target sequence. A variety of different error-correcting codes can be used, many of which have previously been developed for use within the computer industry; however, such error-correcting systems have not typically been used within biological systems. Additional examples of such error-correcting codes are discussed in more detail below.

[0129] In the “incubation” steps as understood herein the respective moi eties or subjects such as probes or oligonucleotide, are brought into contact with each other under conditions well known to the skilled person allowing a specific binding or hybridization reaction, e.g., pH, temperature, salt conditions etc. Such steps may, therefore, be preferably carried out in a liquid environment such as a buffer system which is well known in the art.

[0130] The “removing” steps according to the disclosure may include the washing away of the moi eties or subjects to be removed such as the probes or oligonucleotides by certain conditions, e.g., pH, temperature, salt conditions etc., as known in the art.

[0131] It is understood that in an embodiment of the method according to the present disclosure a plurality of analytes can be encoded in parallel. This requires the use of different sets of analytespecific probes in step (1). The analyte-specific probes of a particular set differ from the analytespecific probes of another set. This means that the analyte-specific probes of set 1 bind to analyte 1, the analyte-specific probes of set 2 binds to analyte 2, the analyte-specific probes of set 3 binds to analyte 3, etc. In this embodiment also the use of different sets of decoding oligonucleotides is required in the methods according to the present disclosure.

[0132] The decoding oligonucleotides of a particular set differ from the decoding oligonucleotides of another set. This means, the decoding oligonucleotides of set 1 bind to the analyte-specific probes of above set 1 of analyte-specific probes, the decoding oligonucleotides of set 2 binds to the analyte-specific probes of above set 2 of analyte-specific probes, the decoding oligonucleotides of set 3 binds to the analyte-specific probes of above set 3 of analyte-specific probes, etc.

[0133] In this embodiment where a plurality of analytes is to be encoded in parallel the different sets of analyte-specific probes may be provided as a premixture of different sets of analyte-specific probes and / or the different sets of decoding oligonucleotides may be provided as a premixture of different sets of decoding oligonucleotides. Each mixture may be contained in a single vial. Alternatively, the different sets of analyte-specific probes and / or the different sets of decoding oligonucleotides may be provided in steps singularly.

[0134] A “kit” is a combination of individual elements useful for carrying out the use and / or method of the disclosure, wherein the elements are optimized for use together in the methods. The kits may also contain additional reagents, chemicals, buffers, reaction vials etc. which may be useful for carrying out the method according to the disclosure. Such kits unify all essential elements required to work the method according to the disclosure, thus minimizing the risk of errors. Therefore, such kits also allow semi-skilled laboratory staff to perform the method according to the present disclosure.

[0135] The term “quencher” or “quencher dye” or “quencher molecule” refers to a dye or an equivalent molecule, such as nucleoside guanosine (G) or 2'-deoxyguanosine (dG), which is capable of reducing the fluorescence of a fluorescent reporter dye or donor dye. A quencher dye may be a fluorescent dye or non-fluorescent dye. When the quencher is a fluorescent dye, its fluorescence wavelength is typically substantially different from that of the reporter dye and the quencher fluorescence is usually not monitored during an assay. Some embodiments of the present disclosure disclose signal oligonucleotides comprising a quencher and / or a quencher in combination with a signal element, and therefore the signal oligonucleotides is not detectable during imaging.

[0136] A “sample” as referred to herein is a composition in liquid or solid form suspected of comprising the analytes to be encoded. A sample described herein may be a biological sample. A sample may comprise biological tissue, biological cells, and / or extracts and / or part of cells, or any combination thereof. The sample may comprise eukaryotic or procaryotic cells. The sample may derive from any kind of animal (including Homo, rat, mouse, mammalia, birds, fish, insects, worms), plant, or fungi. In some embodiments, the sample may comprise a mammalian cell. In some embodiments, the sample may comprise a human cell. The sample can be selected from anyorgan, any tissue, any kind of culture, any specimen taken for diagnostic purposes (e.g. smear, liquid biopsy, tissue biopsy etc.). The sample may comprise DNA and / or RNA. Samples may taken from alive or dead organisms. Cells may not be complete and samples may contain only partial cells. The sample may be frozen, fixed or embedded. In some embodiments, the biological tissue, biological cells, extracts and / or part of cells are fixed. In some embodiments, the analytes are fixed in a permeabilized sample, such as a cell-containing sample.

[0137] The term “cell” as used herein is the smallest unit of life and comprises a number (for example, at least one, at least two, at least 5, at least 10, at least 20) genome elements that can be differentiated. The cell may be dead or alive. The cell may be eukaryotic or prokaryotic. The cell may not be complete and contain only a part of the cell (e.g., due to preparation or fixation of the sample or cell).

[0138] The term “genome” as used herein is a complete identity or a part of this. It can be DNA or RNA.

[0139] The term “transcription” is used herein for a process during which one strand of the genome sequence of the genome element is copied into a complementary RNA (e.g., an mRNA) strand. These single-stranded copies may be independent molecules (not covalently connected to another molecule) but may be connected for a certain time to the genome element by a non- covalently binding (e.g., by hydrogen bonding). The connection can be stabilized by a fixation method (e.g., methanol, formalin etc.).

[0140] The term “biological sample” is defined as a material that is derived from an organism and at least contains detectable nucleic acids, cells or parts of cells. These cells may originate from the same or different organs or even organisms.

[0141] The term “tissue” is used herein for any kind of a sample material that is formed by a certain number of cells of the same or different type with a meaningful structural relationship (or the lack thereof), and thus does comprise genome elements. The term “tissue section” is used herein for a thin section of a tissue favorably done by a cryotome or a microtome.

[0142] An “analyte” according to the disclosure may be any molecule (e.g., a biomolecule) of interest. Sometimes herein the term “analyte” is replaced by “target.” In some embodiments an analyte may comprise a biomolecule (e.g., a protein, a nucleic acid, a biomolecule, a lipid, or any combination thereof). In some embodiments, an analyte may be a nucleic acid (e.g., DNA, PNA, LNA, RNA, or any combination thereof). In some embodiments, an analyte may be a DNA molecule (e.g., genomic DNA, nuclear DNA, circular DNA, mitochondrial DNA, viral DNA, bacterial DNA, extra- or intracellular DNA, or any combination thereof). In some embodiments, an analyte may be an RNA molecule (e.g., mRNA, hnRNA, miRNA, viral RNA, bacterial RNA, extra- or intracellular RNA, circular mRNA, tRNA, siRNA, snRNA, rRNA, or any combinationthereof). In some embodiments, an analyte may be an mRNA (e.g., a transcript). In some embodiments, there may be multiple analytes, e.g., at least two individual nucleic acids.

[0143] In some embodiments, an analyte may be a “coding sequence”, “encoding sequence”, “structural nucleotide sequence”, or “structural nucleic acid molecule” which refers to a nucleotide sequence that is translated into a polypeptide, e.g., via mRNA, when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. A coding sequence can include, but is not limited to, genomic DNA, cDNA, EST, and recombinant nucleotide sequences.

[0144] As used in the present disclosure, “cell”, “cell line”, and “cell culture” can be used interchangeably and all such designations include progeny. Thus, the words “transformants” or “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progenies may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that has the same functionality as screened for in the originally transformed cell are included.

[0145] An “encoding scheme” may describe a set of code words that are associated with the analytes to be detected. Each code word refers to one of the analytes and can be distinguished from all other code words. A code word hereby is a sequence of signs provided by the detection cycles of the method. A sign within a code word is a detectable signal or the absence of a signal. A code word does not need to comprise of all different signals used in the method. The number of signs in a code word is defined by the number of detection cycles.

[0146] An “oligonucleotide” as used herein, refers to a nucleic acid molecule, such as DNA, PNA, LNA or RNA. The length of the oligonucleotides may be within the range of 10-10,000 nucleotides (nt), 10-15,000 nt, 10-10,000 nt, 10-5,000 nt, 10-2,000 nt, 10-1,500 nt, 10-1,000 nt, or 10-800 nt. In some embodiments, the length of oligonucleotides may be within the range of 100-1,500 nt, 100-1,200 nt, 100-1,000 nt, or 100-800 nts. In some embodiments, the length of oligonucleotides may be within the range of 400-1,500 nt, 400-1,200 nt, 400-1,000 nt, or 400-800 nts. The nucleic acid molecule can be fully or partially single-stranded. The oligonucleotides may be linear or may comprise hairpin or loop structures. The oligonucleotides may comprise modifications such as biotin, labeling moieties, blocking moieties, or other modifications.

[0147] “Essentially complementary” means, when referring to two nucleotide sequences, that both sequences can specifically hybridize to each other under stringent conditions, thereby forming a hybrid nucleic acid molecule with a sense and an antisense strand connected to each other via hydrogen bonds (Watson-and-Crick base pairs). “Essentially complementary” includes not only perfect base-pairing along the entire strands, e.g., perfect complementary sequences butalso imperfect complementary sequences which, however, still have the capability to hybridize to each other under stringent conditions. Among experts it is well accepted that an “essentially complementary” sequence has at least 88% sequence identity to a fully or perfectly complementary sequence.

[0148] As disclosed herein, percent sequence identity (e.g., “percent identity,” “percent sequence identity,” or “sequence identity”) describes the similarity between two or more sequences (e.g., a nucleic acid sequence or an amino acid sequence). Sequence similarity calculations may be performed using the BLAST algorithm for sequence alignment, which is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ). Percent sequence identity compares a given sequence to a claimed or described sequence after alignment of the given sequence to be compared (the “Compared Sequence”) with the described or claimed sequence (the “Reference Sequence”). The percent identity is then determined according to the following formula: percent identity = 100*(l -(C / R)) wherein C is the number of differences between the Reference Sequence and the Compared Sequence over the length of alignment between the Reference Sequence and the Compared Sequence, wherein (i) each base or amino acid in the Reference Sequence that does not have a corresponding aligned base or amino acid in the Compared Sequence and (ii) each gap in the Reference Sequence and (iii) each aligned base or amino acid in the Reference Sequence that is different from an aligned base or amino acid in the Compared Sequence, constitutes a difference, and (iv) the alignment has to start at position 1 of the aligned sequences; and R is the number of bases or amino acids in the Reference Sequence over the length of the alignment with the Compared Sequence with any gap created in the Reference Sequence also being counted as a base or amino acid.

[0149] If an alignment exists between the Compared Sequence and the Reference Sequence for which the percent identity as calculated above is about equal to or greater than a specified minimum Percent Identity, then the Compared Sequence has the specified minimum percent identity to the Reference Sequence even though alignments may exist in which the herein above calculated percent identity is less than the specified percent identity.EXAMPLES

[0150] The invention is further illustrated by the following non-limiting examples.EXAMPLE 1 - Cell Fixation of PBMC Cells for Cell Suspension Assays

[0151] This example describes a protocol for the thawing, immobilization and fixation of PBMC cells in pre-assembled and PLL-coated 8-well slides via centrifugation. An overview of the cell fixation protocol is provided in FIG. 1. McCoys Media was warmed in a water bath at 37°C for approx. 20 minutes. PLL (poly-L-lysine) coated coverslips were assembled with an 8-well IBIDIplastic frame with a clamp by clamping each side for 5 minutes. A 4% PFA (paraformaldehyde) solution was prepared. 20mL of the pre-warmed McCoys media was transferred to a 50mL tube. PBMC cells were removed from storage at -80°C and thawed in a 37°C water bath for approximately 2 minutes until only 20% of ice crystals were still visible. The thawed cells were transferred carefully in the 20mL warm McCoys media by pipetting the cells directly into the media. The cells were resuspended carefully and slowly with a lOOOpl pipette. The cell solution was spun down at 300g for 5 minutes. The supernatant was discarded without disturbing the pellet. The cells were resuspended in 5mL PBS (lx) carefully. The cells were spun down at 300-700g for 5 minutes. The cells were resuspended in 1 mL PBS (lx) and transferred to a 1.5mL tube. The cells were spun down at 300g for 5 minutes. The supernatant was carefully discarded without disturbing the pellet. The cells were resuspended in ImL PBS (lx). The cells were stored on ice or at 4°C prior to analysis.

[0152] The total number of cells were counted by a LunaFX7 cell counter under the program bright field cell counting and total cell counting. The cell counting can be inaccurate due to different cell morphologies and sizes so the cell number wase cross-checked with specified cell number from supplier. The cells were then diluted to a cell number of 2x106 cells / mL with PBS. The diluted cell-solution were distributed in the pre-assembled Ibidi slides with 200pl per well for a total number of 4x105 cells / well. The slides were placed in the 3D printed centrifuge inlet and moved in an infinity pattern on the bench to distribute the cells evenly. The slides were spun down at 300g for 5min. The liquid was carefully removed in all wells. 200pl of 4% PFA solution was added to each well and incubated for 2 hours at room temperature. The PFA solution was removed and each well was washed three times with PBS (200pl / well). 200 pl of 70% EtOH was added to each well and incubated for 2 hours at 4°C. The slides were then subjected to one of the following: (i) storage in 70% EtOH at 4°C for up to 7 days until use (likely longer storing possible), (ii) removal of EtOH and dried at room temperature for approximately 10 minutes and storage at - 80°C until use, or (iii) removal of EtOH and proceeded directly with Sudan Black staining (0.2%) of the protocol for spatial transcriptomics. Storing at 4°C (i) or -80°C (ii) may affect transcript detection (e.g., a decrease of -5-10% of transcript detection). The samples were then used for spatial transcriptomics.EXAMPLE 2 - Spatial Transcriptomics of a Cell Suspension Assay

[0153] This example describes spatial transcriptomics analysis of a fixed cell suspension assay. PBMC cells were attached to a slide as described in EXAMPLE 1 and used for a spatial transcriptomics assay to detect transcripts in single cells in the suspension to identify the composition of cells in the PBMC compositions and also to evaluate cell-to-cell interactions.

[0154] Different pre-treatment conditions of the PBMC adhered cell samples were tested including direct entry into the spatial transcriptomics assay, dried at room temperature and then frozen prior to the spatial transcriptomics assay, or storage in ethanol at 4°C prior to the spatial transcriptomics assay. The PBMC were then pre-stained with a violet cell staining or a CFSE cell staining prior to the spatial transcriptomics assay to identify cell populations that were differentially treated. The transcripts within each of the cells were then measured by the spatial transcriptomics assay. The transcripts measured for their gene expression included CD3G, CCR7, CD40LG, CD8A, FOXP3, NCAM1, TRGC1, IL7R, GNLY, PRF1, CD14, FCGR3A, CD1C, LILRA4, TNFRSF17, and MS4A1.

[0155] As shown in FIG. 2, differentially treated cells were distinguished by the violet and CFSE dyes and the spatial transcriptomics assay identified transcripts in each of the single cells withing the cell suspensions. Further shown in FIG. 3, the total transcript counts per tile for each of the tested conditions were comparable for the tested fixation times. FIG. 4 shows the distribution of the gene count and expression per cell and the cell size distribution.

[0156] As shown in FIG. 5, the detected transcripts in each of the cells in the cell suspension successfully identified the complete immune cell repertoire within the PBMC cell population sample. This demonstrates that the spatial transcriptomics assay of the PBMC cell suspension offers highly specific target signal detection and is sensitive to cell-specific gene expression. The spatial transcriptomics assay of the cell suspension enables accurate detection of diverse cell populations while preserving measurement of cell-to-cell interactions. FIG. 6 shows the Uniform Manifold Approximation and Projection (uMAP) identifying each of the cell types present in the PBMC cell suspension. Further shown in FIG. 7A and FIG. 7B, the cell-specific gene expression co-relates with the respective cell populations in the uMAP.EXAMPLE 3 - Spatial Transcriptomics of a Cell Suspension Assay

[0157] This example describes spatial transcriptomics analysis of a cell suspension to evaluate cell to cell interactions in a cell suspension. A subpopulation of cells were exposed to an effector and then exposed to a subpopulation of the cells that were not exposed to an effector to test for differential gene expression resulting from the cell-to-cell interaction in a cell suspension by spatial transcriptomics. This gene expression profile was then compared to cells that were close in proximity, but in which no cell subpopulation was exposed to an effector as a control cell-to- cell interaction in a cell suspension. As shown in FIG. 8, when comparing the cell-to-cell interaction between i) cells that were exposed to an effector and cells that were not exposed to an effector and ii) the control comprising cells that were not exposed to an effector and cells thatwere not exposed to an effector, there was a significant difference in gene expression of the genes measured by spatial transcriptomics.EXAMPLE 4 - Cell Fixation of Non- Adherent Cells for Cell Suspension Assays

[0158] This example describes a protocol for the processing steps for non-adherent cells to subsequently perform a functional interaction assay (e.g., a cell suspension assay). It involves the immobilization and fixation of cells in pre-assembled 8-well Molecular Observation slides.

[0159] All the following reagents are prepared on the day of usage and can be stored at room temperature during the day. The reagents are added in the listed order. When preparing reagents in a bottle, a magnetic stir plate and stir rod for mixing for 1 minute between addition of each component. When preparing reagents in a tube, the tube is inverted and vortexed between addition of each reagent. The reagents warm up to room temperature for 30 minutes.

[0160] The following buffers and solutions are prepared. Buffer PBS (lx): 12,600 pL water and1.400 pL PBS (10X) are combined for a total volume of 14,000 pL. 4% Formaldehyde solution: 1,394 pL water, 176 pL PBS (lOx), and 190 pL formaldehyde are combined for a total volume of 1,760 pL. 70% Ethanol: 1,320 pL water and 3,080 pL ethanol are combined for a total volume of4.400 pL.

[0161] Non-adherent cells are resuspended in Buffer PBS (lx) at a concentration of 2 x 106 cells / mL. If cells were frozen in a DMSO containing medium, the DMSO is removed before seeding the cells on the Molecular Observation slide (e.g., by washing cells in PBS). 200 pL of the cell suspension (containing 4 x 105 cells) are transferred into each well of the Molecular Observation slide. The Molecular Observation slide is placed into the 3D printed centrifuge inlet. The cells are distributed evenly in the wells by moving the Molecular Observation slide vigorously in all planar directions, e.g., moving the slide by following the imaginary line of the number 8 on the bench carefully to avoid breakage and any scratches. The slide is centrifuged for 5 min at 300 x g. The liquid is carefully aspirated from all wells. 200 pL of 4% Formaldehyde solution is added per well and incubated for 2 hours at room temperature. The liquid is aspirated from all wells. Each well is washed three times with 200 pL Buffer PBS (lx) per well. The liquid is carefully aspirated from all wells. 200 pL of 70% Ethanol is added per well and incubated for 2 hours at 4°C. The Molecular Observation slide can be stored and shipped either dried at -80°C or with 70% Ethanol at 4°C.

[0162] For storage at -80°C: the liquid is aspirated from all wells and the Molecular Observation slide is dried for 10 min at room temperature then placed into -80°C freezer overnight. The Molecular Observation slide can be stored up to 2 months at -80°C. The Molecular Observation slide can be transported (e.g., shipped) on dry ice.

[0163] For storage at 4°C: the 70% Ethanol is retained in all wells and stored at 4°C. The Molecular Observation slide can be stored up to 2 weeks at 4°C. The Molecular Observation slide can be transported (e.g., shipped) by filling all wells to the top by adding 300 pL of 70% Ethanol per well (total volume: 500 pL per well), wrapping the Molecular Observation slide (with closed lid) using Parafilm (carefully but tightly without bending or breaking the Molecular Observation slide during wrapping), vacuum-sealing the parafilm-wrapped Molecular Observation slide, and packing the Molecular Observation slide with cool packs to keep the temperature at about 4°C during transport.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method for measuring an interaction between two or more cells in a cell suspension comprising:(i) depositing the two or more cells onto a surface forming the cell suspension on the surface,(ii) performing a spatial analysis of the two or more cells, and(iii) analyzing the spatial analysis to determine the interaction between the two or more cells.

2. The method of claim 1, wherein the surface is a planar surface.

3. The method of claim 1 or claim 2, wherein the surface is planar glass.

4. The method of any one of claims 1-3, wherein the surface is coated with a coating.

5. The method of claim 4, wherein the coating comprises a biocompatible coating.

6. The method of claim 5, wherein the biocompatible coating is poly-L-lysine (PLL), aminosilane, hydrogel, epoxysilane, or any combination thereof.

7. The method of any one of claim 1-6, wherein the spatial analysis comprises spatial transcriptomics.

8. The method of claim 7, wherein the spatial transcriptomics comprises a multiplex method for detecting different analytes in a sample by sequential signal-encoding of said analytes, comprising the steps of:(A) contacting the sample with at least twenty (20) different sets of analyte-specific probes for encoding of at least 20 different analytes, each set of analyte-specific probes interacting with a different analyte, wherein if the analyte is a nucleic acid each set of analyte-specific probes comprises at least five (5) analyte-specific probes which specifically interact with different substructures of the same analyte, each analyte-specific probe comprising:(aa) a binding element (S) that specifically interacts with one of the different analytes to be encoded, and(bb) an identifier element (T) comprising a nucleotide sequence which is unique to the analyte to be encoded (unique identifier sequence),wherein the analyte-specific probes of a particular set of analyte-specific probes differ from the analyte-specific probes of another set of analyte-specific probes in the nucleotide sequence of the identifier element (T), wherein the analyte-specific probes in each set of analyte-specific probes binds to the same analyte and comprises the same nucleotide sequence of the identifier element (T) which is unique to said analyte; and(B) contacting the sample with at least one set of decoding oligonucleotides per analyte, wherein in each set of decoding oligonucleotides for an individual analyte each decoding oligonucleotide comprises:(aa) an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set, and(bb) a translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide; wherein the decoding oligonucleotides of a set for an individual analyte differ from the decoding oligonucleotides of another set for a different analyte in the first connect element (t); and(C) contacting the sample with at least a set of signal oligonucleotides, each signal oligonucleotide comprising:(aa) a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of a translator element (c) comprised in a decoding oligonucleotide, and(bb) a signal element,(D) detecting the signal caused by the signal element;(E) selectively removing the decoding oligonucleotides and signal oligonucleotides from the sample, thereby essentially maintaining the specific binding of the analyte-specific probes to the analytes to be encoded;(F) performing at least three (3) further cycles comprising steps B) to E) to generate an encoding scheme with a code word per analyte, wherein in particular the last cycle may stop with step (D).

9. The method of claim 8, wherein the analyte comprises a nucleic acid.

10. The method of claim 9, wherein the nucleic acid is an mRNA molecule.

11. The method of any one of claims 1-10, wherein the two or more cells comprise cells dissociated from tissue.

12. The method of any one of claims 1-11, wherein the two or more cells comprise nonadherent cells.

13. The method of any one of claims 1-12, wherein the two or more cells comprise adherent cells.

14. The method of any one of claims 1-13, wherein the two or more cells comprise a rare cell type or a population of cells comprising less than 20 of a cell type.

15. The method of any one of claims 1-14, wherein the two or more cells comprise cells that have been labeled with one or more dyes.

16. The method of any one of claims 1-15, wherein the two or more cells comprise live cells.

17. The method of any one of claims 1-16, wherein the two or more cells comprise dead cells.

18. The method of any one of claims 1-17, wherein the two or more cells comprise cells that interact with at least one cell in the cell suspension.

19. The method of any one of claims 1-18, wherein the two or more cells comprise an additional reagent that induces interactions between cells in the cell suspension.

20. The method of any one of claims 1-19, wherein the two or more cells comprise an antibody.

21. The method of any one of claims 1-20, wherein the two or more cells comprise tumor cells and immune cells.

22. The method of any one of claims 1-21, wherein the two or more cells comprise a pharmaceutical molecule.

23. The method of any one of claims 1-22, wherein the two or more cells comprise an engineered or transfected cell.

24. The method according to any one of the preceding claims, comprising at least one of (A) imaging at least a portion of a sample comprising the at least two or more cells, (B) using an optical imaging technique, (C) using a fluorescence imaging technique, (D) using a multi-colorfluorescence imaging technique, and (E) super-resolution fluorescence imaging technique, in particular to thereby determine a signal caused by a signal element and / or the binding of signal oligonucleotides to decoding oligonucleotides, interacting with the corresponding analyte probes, bound to the respective analyte.

25. The method according to any one of the preceding claims, wherein a coded fluorescence patterns is assigned to a plurality of target analytes in a cell.

26. The method according to any one of the preceding claims, wherein one or plural cells are subjected to a plurality of detection rounds, each detection round in particular comprising at least one of (A) contacting the cell to representatives to a same of at least two populations of tagged fluorescence moieties and (B) removing the fluorescent moieties after a single excitation event27. The method according to any one of the preceding claims, wherein (A) imaging is performed to thereby obtain an image of two or more cells in a sample, and (B) an analyte is assigned to a position in said image.

28. The method according to claim 26, wherein (A) a fluorescence pattern is assigned to an analyte, (B) the fluorescence pattern is observed at the position in the image, and (C) the analyte is assigned to the position.

29. The method according to claim 27, wherein observing the fluorescence pattern comprises repeating steps of (A) labeling a position using a fluorophore tagged oligo drawn from a re- accessible pool, (B) performing a single excitation at the position in the image, and (C) contacting the analyte to a denaturant.

30. The method according to claim 28, wherein observing the fluorescence pattern comprises repeating steps of (A) labeling the position using a fluorophore tag-recruiting bridging oligo drawn from a re-accessible pool, (B) performing a single excitation at the position in the image, and (C) contacting the analyte to a denaturant.

31. The method according to any one of the preceding claims, wherein an optical multiplexing system is used configured to carry out the method and comprising at least one of (A) a reaction vessel receiving, carrying, and / or supporting the one or plural cells; (B) a detection unit comprising a microscope, in particular a fluorescence microscope; (C) a camera; and (D) a liquid handling device.

32. A method of assaying for a cell interaction, comprisingcontacting a first population of cells to an interaction candidate pool, depositing the first population of cells on a surface, performing a spatial analysis on the first population of cells, identifying an interaction between a first cell of the first cell population and a first interaction candidate of the interaction candidate pool, and identifying the first cell using spatial analysis.

33. The method of claim 32, wherein contacting comprises co-culturing the first population of cells and the interaction candidate pool.

34. The method according to any one of claims 32 to 33, wherein contacting comprises agitating the first population of cells so as to disrupt weak interactions with the interaction candidate pool.

35. The method according to any one of claims 32 to 34, wherein the first population of cells comprises a plurality of cell types.

36. The method according to any one of claims 32 to 35, wherein the first population of cells comprises tumor cells.

37. The method according to any one of claims 32 to 36, wherein the first population of cells comprises PBMCs.

38. The method according to any one of claims 32 to 37, wherein the first population of cells comprises stem cells.

39. The method according to any one of claims 32 to 38, wherein the first population of cells comprises a eukaryotic pathogen.

40. The method according to any one of claims 32 to 39, wherein the first population of cells comprises a prokaryotic pathogen.

41. The method according to any one of claims 32 to 40, wherein the first population of cells comprises cells from a disaggregated tissue.

42. The method according to any one of claims 32 to 41, wherein the first population of cells comprises cells from a fluid sample.

43. The method according to any one of claims 32 to 42, wherein the interaction candidate pool comprises a second cell population.

44. The method of claim 43, wherein the second cell population comprises a plurality of cell types.

45. The method according to any one of claims 43 to 44, wherein the second cell population comprises tumor cells.

46. The method according to any one of claims 43 to 45, wherein the second cell population comprises PBMCs.

47. The method according to any one of claims 43 to 46, wherein the second cell population comprises stem cells.

48. The method according to any one of claims 43 to 47, wherein the second cell population comprises a eukaryotic pathogen.

49. The method according to any one of claims 43 to 48, wherein the second cell population comprises a prokaryotic pathogen.

50. The method according to any one of claims 43 to 49, wherein the second cell population comprises cells from a disaggregated tissue.

51. The method according to any one of claims 43 to 50, wherein the second cell population comprises cells from a fluid sample.

52. The method according to any one of claims 32 to 51, wherein the interaction candidate pool comprises a virus population.

53. The method according to any one of claims 32 to 52, wherein the interaction candidate pool comprises an antibody.

54. . The method according to any one of claims 32 to 53, wherein the interaction candidate pool comprises a small molecule.

55. The method according to any one of claims 32 to 54, wherein the surface comprises surface coatings and / or positional oligo markers.

56. The method according to any one of claims 32 to 55, wherein the spatial analysis comprises fluorescence in situ hybridization.

57. The method according to any one of claims 32 to 56, wherein the spatial analysis comprises hybridizing a probe to a nucleic acid of the first cell.

58. The method of claim 57, wherein the probe is a fluorophore labeled probe.

59. The method according to any one of claims 57 to 58, wherein the probe is an oligo labeled probe.

60. The method according to any one of claims 57 to 59, wherein the probe comprises an analyte identifying oligo.

61. The method according to any one of claims 32 to 60, wherein the spatial analysis comprises contacting a probe to a protein of the first cell.

62. The method of claim 61, wherein the probe is a fluorophore labeled antibody.

63. The method according to any one of claims 61 to 62, wherein the probe is an oligo labeled antibody.

64. The method according to any one of claims 61 to 63, wherein the probe comprises an analyte identifying oligo.

65. The method according to any one of claims 32 to 64, wherein the spatial analysis comprises assaying for at least 20 distinct transcripts in the first cell.

66. The method according to any one of claims 32 to 65, wherein the spatial analysis comprises assaying for at least 20 distinct transcripts in cells of the first cell population.

67. The method according to any one of claims 32 to 66, wherein the spatial analysis comprises assaying for at least 1,000 distinct transcripts in the first cell.

68. The method according to any one of claims 32 to 67, wherein the spatial analysis comprises assaying for at least 1,000 distinct transcripts in cells of the first cell population.

69. The method according to any one of claims 32 to 68, wherein the spatial analysis comprises assaying for at least 20 distinct epitopes in the first cell.

70. The method according to any one of claims 32 to 69, wherein the spatial analysis comprises assaying for at least 20 distinct epitopes in cells of the first cell population.

71. The method according to any one of claims 32 to 70, wherein the spatial analysis comprises generating position-tagged sequence information for the first cell.

72. The method according to any one of claims 32 to 71, wherein the spatial analysis comprises generating position-tagged sequence information for the first cell population.

73. The method according to any one of claims 32 to 72, wherein the spatial analysis comprises generating sequence information for at least 20 distinct transcripts in the first cell.

74. The method according to any one of claims 32 to 73, wherein the spatial analysis comprises generating sequence information for at least 1,000 distinct transcripts in the first cell.

75. The method according to any one of claims 32 to 74, wherein the spatial analysis comprises generating sequence information for at least 20 distinct loci in the first cell.

76. The method according to any one of claims 32 to 75, wherein the spatial analysis comprises generating sequence information for at least 1,000 distinct loci in the first cell.

77. The method according to any one of claims 32 to 76, wherein identifying an interaction between a first cell of the first cell population and a first interaction candidate comprises localizing the first cell and the first interaction candidate within physical proximity to one another.

78. The method of claim 77, wherein localizing comprises visualizing the first cell and the first interaction partner within physical proximity to one another in an image of the surface.

79. The method according to any one of claims 77 to 78, wherein localizing comprises localizing a signal from a probe that identifies the first cell and a probe that identifies the first interaction partner within physical proximity to one another.

80. The method according to any one of claims 32 to 79, wherein identifying the first cell using the spatial analysis comprises identifying a signal from a probe that is specific to the first cell.

81. The method according to any one of claims 32 to 80, wherein identifying the first cell using the spatial analysis comprises identifying a signal from a probe that identifies the first cell.

82. The method according to any one of claims 32 to 81, wherein identifying the first cell using the spatial analysis comprises identifying a pattern of probe signals that is specific to the first cell.

83. The method according to any one of claims 32 to 82, wherein identifying the first cell using the spatial analysis comprises identifying a pattern of probe signals that identifies the first cell.

84. The method of claim 83, comprising comparing the first cell pattern of probe signals to a second cell pattern of probe signals.

85. The method according to any one of claims 83 to 84, wherein the second cell pattern of probe signals is obtained from a second cell that is not in proximity to an interacting partner.

86. The method according to any one of claims 83 to 85, wherein the second cell pattern of probe signals is obtained from a second cell that is in proximity to an interacting partner.

87. The method according to any one of claims 83 to 86, wherein the second cell pattern of probe signals is obtained from a second cell that is of a common cell type with the first cell.

88. The method according to any one of claims 32 to 87, wherein the method is untargeted.

89. The method according to any one of claims 32 to 88, wherein the method identifies at least 10 distinct categories of cells involved in interactions.

90. The method according to any one of claims 32 to 89, wherein the method identifies at least 20 distinct categories of cells involved in interactions.

91. The method according to any one of claims 32 to 90, wherein the method identifies at least 50 distinct categories of cells involved in interactions.

92. A method of characterizing a cell interaction, comprising subjecting a cell population to interaction conditions such that a first cell of a first cell type of the cell population interacts with a binding partner, and generating spatial analysis data for the cell population.

93. The method of claim 92, wherein generating spatial analysis data for the cell population comprises [HERE fish, antibodies, analyte tagging probes, sequencing, number of analytes assayed.

94. The method according to any one of claims 92 to 93, wherein generating spatial analysis data for the cell population comprises obtaining transcript location information for at least 20 transcripts.

95. The method according to any one of claims 92 to 94, wherein generating spatial analysis data for the cell population comprises obtaining transcript location information for at least 50 transcripts.

96. The method according to any one of claims 92 to 95, wherein generating spatial analysis data for the cell population comprises obtaining transcript location information for at least 100 transcripts.

97. The method according to any one of claims 92 to 96, wherein generating spatial analysis data for the cell population comprises obtaining transcript location information for at least 200 transcripts.

98. The method according to any one of claims 92 to 97, wherein generating spatial analysis data for the cell population comprises obtaining transcript location information for at least 500 transcripts.

99. The method according to any one of claims 92 to 98, wherein generating spatial analysis data for the cell population comprises obtaining transcript location information for at least 1000 transcripts.

100. The method according to any one of claims 92 to 99, wherein generating spatial analysis data for the cell population comprises obtaining transcript location information for at least 2000 transcripts.

101. The method according to any one of claims 92 to 100, wherein generating spatial analysis data for the cell population comprises obtaining transcript location information for at least 5000 transcripts.

102. The method according to any one of claims 92 to 101, wherein generating spatial analysis data for the cell population comprises obtaining transcript location information for at least 10,000 transcripts.

103. The method according to any one of claims 92 to 102, wherein generating spatial analysis data for the cell population comprises obtaining transcript location information for at least 20,000 transcripts.

104. The method according to any one of claims 92 to 103, comprising comparing the spatial analysis data for the first cell of the first cell type to a second cell of the first cell type, wherein the second cell of the first cell type does not interact with the binding partner.

105. The method according to any one of claims 92 to 104, comprising identifying binding partner impact on the first cell spatial analysis data relative to the second cell of the first cell type.

106. The method according to any one of claims 92 to 105, comprising comparing the spatial analysis data for the first cell of the first cell type to a reference spatial analysis data set for the first cell type.

107. The method according to any one of claims 92 to 106, comprising identifying binding partner impact on the first cell spatial analysis data relative to the reference spatial analysis data set for the first cell type.

108. The method of claim 107, wherein the reference spatial analysis data set for the first cell type is used to identify the first cell of the first cell type.

109. The method according to any one of claims 107 to 108, wherein the reference spatial analysis data set for the first cell type is generated from a first cell type that is not interacting with the binding partner.

110. The method according to any one of claims 92 to 109, wherein generating spatial analysis data for the cell population comprises generating spatial analysis data for the binding partner.

111. The method of claim 110, comprising using the spatial analysis data for the binding partner to identify the binding partner.

112. The method according to any one of claims 92 to 111, wherein the method is untargeted.

113. The method according to any one of claims 92 to 112, wherein the method identifies at least 10 distinct categories of cells involved in interactions.

114. The method according to any one of claims 92 to 113, wherein the method identifies at least 20 distinct categories of cells involved in interactions.

115. The method according to any one of claims 92 to 114, wherein the method identifies at least 50 distinct categories of cells involved in interactions.

116. A method of identifying interacting cells in a cell population, comprising fixing the cell population so that its cells are held in place, performing a spatial analysis on the cell population, identifying cells of the cell population through the spatial analysis, and identifying cells in close physical proximity as interacting.

117. The method of claim 116, wherein the cell population comprises cells that are treated to be disaggregated prior to the fixing.

118. The method according to any one of claims 116 to 117, wherein the cell population comprises cells obtained from a liquid sample.

119. The method according to any one of claims 116 to 118, wherein fixing the cell population comprises stabilizing the cell population in a matrix.

120. The method according to any one of claims 116 to 119, wherein fixing the cell population comprises affixing the cell population to a surface.

121. The method of claim 120, wherein the surface comprises an adherent.

122. The method according to any one of claims 120 to 121, wherein the surface comprises an oligo field.

123. The method of claim 122, wherein the oligo field comprises oligos conveying position indicative information.

124. The method according to any one of claims 116 to 123, wherein the spatial analysis comprises obtaining transcriptional information for at least some cells of the cell population.

125. The method of claim 124, wherein obtaining transcriptional information comprises hybridizing analyte probes to transcripts of at least some cells of the cell population.

126. The method of claim 125, wherein the analyte probes comprise analyte indicative fluorophore labels.

127. The method according to any one of claims 125 to 126, wherein the analyte probes comprise probe indicative fluorophore labels, such that a first analyte probe comprises a first fluorophore label and a second analyte probe comprises a second fluorophore label.

128. The method according to any one of claims 125 to 127, wherein the analyte probes comprise analyte indictive tags, such that a plurality of analyte probes targeting a common analyte share a common tag.

129. The method of claim 128, wherein the common tag does not distinguish among the plurality of analyte probes targeting the common analyte.

130. The method of claim 129, wherein the common tag does not identify any individual probe among the plurality of analyte probes targeting the common analyte.

131. The method according to any one of claims 127 to 130, comprising hybridizing a first plurality of decoding probes to the sample, wherein the first plurality of decoding probes comprises a decoding probe having an analyte indicative tag binding region and a first signal probe binding region, so as to anneal to analyte indictive tags.

132. The method of claim 131, wherein a first decoding probe of the decoding probes anneals to a first analyte indicative tag of the analyte indicative tags at a melting temperature that is lower than a melting temperature of an interaction between a first analyte probe and a first transcript.

133. The method of claim 132, comprising annealing a first signal probe to the first decoding probe, wherein the first signal probe comprises a first fluorophore.

134. The method of claim 133, comprising removing the first decoding probe, and hybridizing a second plurality of decoding probes to the sample, wherein the second plurality of decoding probes comprises a decoding probe having an analyte indicative tag binding region and a second signal probe binding region, so as to anneal to analyte indictive tags.

135. The method of claim 134, wherein a second decoding probe of the decoding probes anneals to a first analyte indicative tag of the analyte indicative tags at a melting temperature that is lower than a melting temperature of an interaction between a first analyte probe and a first transcript.

136. The method according to any one of claims 132 to 136, comprising annealing a second signal probe to the second decoding probe, wherein the second signal probe comprises a second fluorophore.

137. The method of claim 136, wherein an order of first fluorophore signals and second fluorophore signals specifies the transcript.

138. The method according to any one of claims 127 to 137, wherein an order of first fluorophore signals and second fluorophore signals is specified by an order of addition of first decoding probes and second decoding probes.

139. The method of claim 138, wherein an order of addition of first decoding probes and second decoding probes is not specified by a sequence of the first analyte specific tag.

140. The method of claim 139, wherein an order of addition of first decoding probes and second decoding probes is specified by a user.

141. The method according to any one of claims 116 to 140, wherein obtaining transcriptional information comprises generating a sequencing library that preserves transcript positional information, sequencing the library, and assigning sequences to positions indicated by the transcript position information.

142. The method of claim 141, wherein generating a sequencing library that preserves transcript positional information comprises tagging transcripts of the cell population using position indicative oligos.

143. The method according to any one of claims 116 to 143, wherein the spatial analysis comprises obtaining protein information for at least some cells of the cell population144. The method of claim 143, wherein the protein information comprises cell surface protein information.

145. The method according to any one of claims 116 to 144, wherein identifying cells of the cell population through the spatial analysis comprises assigning a plurality of localized transcript signals to a common cell, and assigning that cell to a cell category having a transcript accumulation pattern that does not differ significantly from the plurality of localized transcript signals of the cell category.

146. The method according to any one of claims 116 to 146, wherein assigning a plurality of localized transcript signals to a common cell comprises identifying a cell border of the common cell using transmission microscopy.

147. The method of claim 146, wherein identifying cells of the cell population through the spatial analysis comprises obtaining surface protein data for cells of the cell population.

148. The method according to any one of claims 116 to 147, wherein identifying cells in close physical proximity as interacting comprises identifying a point of contact between cells in close physical proximity.

149. The method according to any one of claims 116 to 148, wherein identifying cells in close physical proximity as interacting comprises identifying a colinear border between cells in close physical proximity.

150. The method according to any one of claims 116 to 150, wherein identifying cells in close physical proximity as interacting comprises identifying an enrichment of occurrence of the cells in close physical proximity relative to an expected occurrence given the cell population.

151. The method according to any one of claims 116 to 151, wherein the method is untargeted.

152. The method according to any one of claims 116 to 152, wherein the method identifies at least 10 distinct categories of cells involved in interactions.

153. The method according to any one of claims 116 to 152, wherein the method identifies at least 20 distinct categories of cells involved in interactions.

154. The method according to any one of claims 116 to 153, wherein the method identifies at least 50 distinct categories of cells involved in interactions.

155. A composition comprising a first cell of a first cell type, a second cell of a second cell type in physical contact with the first cell of the first cell type, a surface to which at least one of the first cell and the second cell is affixed, and a population of analyte probes comprising a first set of analyte probes that independently target a first analyte and share a common first analyte tag, a second set of analyte probes that independently target a second analyte and share a common second analyte tag.

156. The composition of claim 155, wherein the composition comprises at least 20 cell types.

157. The composition according to any one of claims 155 to 156, wherein the composition comprises at least 500 cells.

158. The composition according to any one of claims 155 to 157, wherein the first set of analyte probes comprises oligonucleotide probes.

159. The composition according to any one of claims 155 to 158, wherein the first analyte tag does not identify a specific probe of the first set of analyte probes.

160. The composition according to any one of claims 155 to 159, wherein the population of analyte probes comprises at least 10 sets of analyte probes.

161. The composition according to any one of claims 155 to 160, wherein the population of analyte probes comprises at least 20 sets of analyte probes.

162. The composition according to any one of claims 155 to 161, wherein the population of analyte probes comprises at least 50 sets of analyte probes.

163. The composition according to any one of claims 155 to 162, wherein the population of analyte probes comprises at least 100 sets of analyte probes.

164. The composition according to any one of claims 155 to 163, wherein the population of analyte probes comprises at least 200 sets of analyte probes.

165. The composition of claim 158, wherein the population of analyte probes comprises at least 500 sets of analyte probes.

166. The composition according to any one of claims 155 to 165, wherein the population of analyte probes comprises at least 1000 sets of analyte probes.

167. The composition according to any one of claims 155 to 166, wherein the first analyte is a nucleic acid.

168. The composition according to any one of claims 155 to 167, wherein the nucleic acid is an RNA molecule.

169. The composition of claim 168, wherein the nucleic acid is a DNA locus.

170. The composition according to any one of claims 155 to 169, comprising a first set of decoding probes.

171. The composition of claim 170, wherein a decoding probe of the first set of decoding robes comprises a first analyte tag binding region and a first signal probe binding region.

172. The composition according to any one of claims 170 to 171, wherein the first signal probe binding region is annealed to a first signal probe comprising a first fluorophore.

173. The composition of claim 172, wherein a decoding probe of the second set of decoding robes comprises a second analyte tag binding region and a first signal probe binding region.

174. The composition of claim 173, wherein the first signal probe binding region is annealed to a first signal probe comprising a first fluorophore.

175. The composition of claim 174, wherein a decoding probe of a third set of decoding robes comprises a third analyte tag binding region and a second signal probe binding region.

176. The composition according to any one of claims 173 to 175, wherein the second signal probe binding region is annealed to a second signal probe comprising a second fluorophore.

177. The composition according to any one of claims 155 to 176, wherein the composition comprises no more than 2 types of fluorophores.

178. The composition according to any one of claims 155 to 177, wherein the composition comprises no more than 3 types of fluorophores.

179. The composition according to any one of claims 155 to 178, wherein the composition comprises no more than 10 types of fluorophores.

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