Chemical composition and method of use thereof

Abstract

The present invention provides compositions and methods for detecting and identifying target nucleic acids in tissue samples using a fluorescent probe. [Solution] This disclosure provides a composition and method for detecting and identifying a target nucleic acid in a tissue sample using a fluorescent probe, characterized in that the probe comprises a target-binding domain and a barcode domain.

Description

[Technical Field]

[0001] [Cross-reference to related applications] This application claims priority to and benefit from U.S. Provisional Patent Application No. 63 / 078,965, filed on 16 September 2020. The contents of the said patent application are incorporated herein by reference in their entirety for any purpose. [Background technology]

[0002] [Background of the Invention] While various methods exist for detecting nucleic acids and proteins in biological samples, there remains a need for improved, accurate, rapid, and highly sensitive multiplexed detection, identification, and quantification of target nucleic acids and proteins in biological samples. Specifically, there is a need for the ability to detect the abundance and spatial arrangement of specific nucleic acids and target proteins in tissue samples while maintaining their original form. This disclosure addresses this need. [Overview of the Initiative]

[0003] This disclosure provides a method for determining the abundance and spatial arrangement of at least two target analytes in a biological sample, wherein the biological sample is prepared by i) incubating the biological sample with a solution containing a plurality of ISH probes, thereby contacting the biological sample with at least one nucleic acid probe, wherein the solution contains at least two ISH probes, at least one of which contains a unique target-binding domain that binds to one of at least two target analytes and a unique barcode domain specific to the target analyte, the barcode domain containing at least one attachment site, and ii) washing the biological sample; the method is, a) A step of contacting a prepared biological sample with a plurality of reporter probes, each of which reporter probes includes at least one detectable label, thereby hybridizing the reporter probe to the attachment area of ​​the barcode region of at least one ISH probe hybridized to a target analyte in the biological sample; b) A step of removing reporter probes that did not hybridize from the biological sample; c) A step of recording the attributes and spatial arrangement of the detectable labels of the hybridized reporter probes; d) A step of removing the detectable labels of the hybridized reporter probes; and e) A step of repeating steps (a) to (d) until each attachment position within the barcode domain of the ISH probe hybridized to the target analyte is hybridized to a reporter probe containing at least one detectable label, thereby determining the abundance and spatial arrangement of at least two target analytes in the biological sample based on the recorded sequence (linear order) of the detectable labels.

[0004] In some aspects of the method of the present disclosure, at least two target analytes are target nucleic acid molecules, the target-binding domain is a single-stranded polynucleotide containing a nucleic acid sequence complementary to the target nucleic acid, the target-binding domain is about 35 to about 40 nucleotides long, the target-binding domain contains D-DNA, and the barcode domain is a single-stranded polynucleotide containing at least one attachment region, each of which attachment regions contains about one attachment sequence, each of which attachment sequences is about 14 nucleotides long, each of which attachment sequences is distinct, and the barcode domain contains L-DNA.

[0005] In some aspects of the method of the present disclosure, at least two target analytes are target protein molecules, and the target-binding domain comprises a protein, preferably an antibody or antigen-binding fragment that specifically binds to the target protein molecule.

[0006] In some aspects of the method of the present disclosure, the barcode domain includes i) at least two, ii) at least three, iii) at least four, or iv) at least five attachment regions.

[0007] In some aspects of the method of the present disclosure, the solution comprises at least one negative ISH probe designed not to specifically bind to any target analyte in the biological sample, preferably the ISH probe comprising at least one External RNA Control Consortium (ERCC) evaluation sequence or its complement. In some aspects of the method of the present disclosure, the negative ISH probe is used to determine the level of background noise in the biological sample.

[0008] In some aspects of the methods of this disclosure, the reporter probe comprises L-DNA.

[0009] In some aspects of the method of the present disclosure, the reporter probe comprises a first nucleic acid molecule comprising a first domain, a second domain, and a photocleavable linker positioned between the first and second domains, wherein the second domain of the first nucleic acid molecule hybridizes to about six second nucleic acid molecules, each second nucleic acid molecule comprising a first domain, a second domain, and a photocleavable linker positioned between the first and second domains, wherein the first domain of each second nucleic acid molecule hybridizes to the second domain of the first nucleic acid molecule, and the second domain of each second nucleic acid molecule hybridizes to about five third nucleic acid molecules, each third nucleic acid molecule comprising at least one detectable label, and the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule each comprising L-DNA.

[0010] In some aspects of the method of this disclosure, at least one detectable label is a fluorescent portion.

[0011] In some embodiments of the method of the present disclosure, prior to step (a), the method further comprises: i) incubating the biological sample in a sulfo-NHS acetate blocking solution for about 15 minutes; ii) washing the biological sample with a reporter wash buffer; iii) incubating the biological sample in a autofluorescence suppression buffer and / or irradiating the biological sample with blue light and / or UV light to thereby quench the autofluorescence of the sample via photobleaching; and iv) washing the biological sample with a reporter wash buffer.

[0012] In some embodiments of the method of the present invention, step (a) comprises incubating the biological sample with a solution containing DEPC-treated water, a reporter probe at a concentration of 5 nM, 8.75×SSPE solution, 0.5% Tween™-20, and optionally 0.1% RNase inhibitor for at least about 15 minutes.

[0013] In some embodiments of the method of the present disclosure, step (b) comprises washing the biological sample with a reporter wash buffer.

[0014] In some embodiments of the method of the present disclosure, step (c) comprises: i) immersing the biological sample in an imaging buffer; and ii) imaging the biological sample to record the attributes and spatial arrangement of the detectable label of the hybridized reporter probe.

[0015] In some embodiments of the method of the present disclosure, step (d) comprises performing at least one or both of: i) irradiating the biological sample with UV light sufficient to cleave the photocleavable linker moiety in the hybridized reporter probe and washing the biological sample with a stripping buffer; and optionally, step (d) further comprises: iii) immersing the biological sample in an imaging buffer; iv) imaging the sample to confirm that no detectable label remains.

[0016] In some aspects of the method of the present disclosure, the method further includes performing a morphological scan of a biological sample to determine one or more regions of interest. Preferably, performing the morphological scan includes: i) staining the biological sample with a membrane-specific fluorescent staining solution and imaging the biological sample to identify the spatial arrangement of cell membranes within the sample; ii) staining the biological sample with a nucleus-specific fluorescent staining solution and imaging the biological sample to identify the spatial arrangement of cell nuclei in the sample; and iii) performing cell segmentation.

[0017] In some aspects of the method of the present disclosure, before contacting the biological sample with at least one nucleic acid probe, the biological sample is further prepared by: aa) mounting the biological sample on a functionalized microscope slide, thereby preparing the mounted biological sample, where the biological sample is a formalin-fixed paraffin-embedded (FFPE) microtome section; bb) baking the mounted biological sample; cc) deparaffinizing the mounted biological sample; dd) performing a target activation reaction on the mounted biological sample; ee) permeating the mounted biological sample; ff) applying at least one reference marker to the mounted biological sample; and gg) fixing the mounted biological sample.

[0018] In some aspects of the method of the present disclosure, after step (ii), the method further includes assembling the mounted biological sample into a flow cell.

[0019] In some aspects of the method of the present disclosure, the functionalized microscope slide is a positively charged microscope slide. Preferably, the functionalized microscope slide is a microscope slide functionalized with (3-aminopropyl)trimethoxysilane (APTMS).

[0020] In some aspects of the method of the present disclosure, the biological sample is an FFPE microtome section of a human tissue sample.

[0021] In some aspects of the method disclosed herein, step (bb) includes calcining the attached biological sample at approximately 60°C for approximately 1 hour.

[0022] In some aspects of the method of the present disclosure, step (cc) includes i) incubating the mounted biological sample in a first xylene solution for about 5 minutes; ii) incubating the mounted biological sample in a second xylene solution for about 5 minutes; iii) incubating the mounted biological sample in a first 100% ethanol solution for about 2 minutes; iv) incubating the mounted biological sample in a second 100% ethanol solution for about 2 minutes; and v) drying the mounted biological sample at about 60°C for about 5 minutes.

[0023] In some aspects of the method of the present disclosure, step (dd) includes i) incubating the attached biological sample in a target activation solution at about 100°C, ii) incubating the attached biological sample in DEPC-treated water for about 15 seconds, iii) incubating the attached biological sample in a 100% ethanol solution for about 3 minutes, and iv) drying the attached biological sample.

[0024] In some aspects of the method disclosed herein, the attached biological sample is incubated in a Target Retrieval Solution for the time specified in Table 1.

[0025] In some aspects of the method of this disclosure, the target retrieval solution comprises a TRIS and EDTA solution and has a pH of about 9.

[0026] In some aspects of the method of the present disclosure, step (ee) includes i) incubating the mounted biological sample in a proteinase solution at about 40°C, wherein the proteinase solution comprises protease K; ii) washing the biological sample with a first aliquot of DEPC-treated water; and iii) washing the biological sample with a second aliquot of DEPC-treated water.

[0027] In some aspects of the method disclosed herein, the attached biological sample is incubated in a proteinase K solution for the time specified in Table 2.

[0028] In some aspects of the method of the present disclosure, step (ff) includes i) incubating the mounted biological sample in a solution containing at least one reference marker for about 5 minutes at about room temperature, wherein the solution containing at least one reference marker is a solution containing carboxylated microspheres stained red, yellow, blue and / or green at a concentration of about 0.0005% to about 0.003% in 2×SSCT solution, and ii) washing the mounted biological sample with 1×PBS.

[0029] In some aspects of the method of the present disclosure, step (gg) includes i) incubating the mounted biological sample in 10% NBF for about 1 minute; ii) incubating the mounted biological sample in a first Tris-glycine buffer solution for about 5 minutes; iii) incubating the mounted biological sample in a second Tris-glycine buffer solution for about 5 minutes; and iv) incubating the mounted biological sample in 1×PBS for about 5 minutes.

[0030] In some aspects of the method of the present disclosure, the method further comprises incubating the attached biological sample in a blocking solution after step (gg), wherein the incubation of the attached biological sample in the blocking solution comprises i) incubating the attached biological sample in a sulfo-NHS-acetic acid / Tween20™ solution for about 15 minutes, wherein the sulfo-NHS-acetic acid / Tween20 solution comprises about 100 mM sulfo-NHS-acetic acid and about 0.5% Tween20 in about 100 mM sodium phosphate pH 8.0, and ii) incubating the attached biological sample in 1×PBS for about 5 minutes.

[0031] In some aspects of the method of the present disclosure, incubation of a fitted biological sample with a solution containing multiple ISH probes includes incubating the fitted biological sample with a solution containing multiple ISH probes at approximately 37°C for approximately 16 to approximately 18 hours, thereby hybridizing at least one ISH probe to a target analyte in the biological sample.

[0032] In some aspects of the method of the present disclosure, washing a biological sample includes: i) incubating the attached biological sample with a first 2×SSC solution; ii) incubating the attached biological sample in a first formamide solution; iii) incubating the attached biological sample with a second formamide solution; iv) incubating the attached biological sample with a second 2×SSC solution; and v) incubating the attached biological sample with a third 2×SSC solution.

[0033] Any of the embodiments described above or herein can be combined with any other embodiments.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which this disclosure belongs. In this specification, singular forms include plural forms unless the context clearly indicates otherwise; for example, the terms “a,” “an,” and “the” are interpreted as singular or plural, and the term “or” is interpreted as comprehensive. For example, “an element” means one or more elements. Throughout this specification, it will be understood that “comprising” or variations thereof, such as “comprises” or “comprising,” means to encompass the element, integer or process, or group of elements, integers or processes, mentioned, but not to exclude any other group of elements, integers or processes. The term "approximately" can be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the mentioned figure. Unless otherwise evident from the context, all figures provided herein are modified by the term "approximately."

[0035] Methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, but preferred methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated herein by reference in their entirety. References cited herein are not considered prior art to the inventions described in the claims. In case of any inconsistency, this specification shall prevail, including in definitions. In addition, materials, methods, and examples are illustrative and not intended to be limiting. Other features and advantages of this disclosure will become apparent from the detailed description below and the claims. [Brief explanation of the drawing]

[0036] The above and further features will be more clearly understood from the following detailed description in conjunction with the attached drawings.

[0037] [Figure 1] Figure 1 is a schematic diagram of an exemplary in situ hybridization (ISH) probe of this disclosure. [Figure 2] Figure 2 is a schematic diagram of an exemplary reporter probe of the present disclosure. [Figure 3] Figures 3A, 3B, 3C, 3D, 3E, 3F, and 3H are schematic diagrams illustrating the steps of a method for detecting the abundance and spatial arrangement of multiple target nucleic acids in a biological sample. [Figure 4] Figure 4 shows a series of graphs comparing the abundance of RNA target analytes in various cells measured using the methods of this disclosure and standard RNA-seq methods. [Figure 5] Figure 5 shows images of individual target analytes detected in a biological sample containing MDA-MB-468 cells using the method of this disclosure. Figure 5 also shows the quantification of the number of transcripts per cell analyzed. [Figure 6A] Figure 6A shows images of individual target analytes detected in melanoma FFPE tissue samples (specimens) using the method of this disclosure. [Figure 6B] Figure 6B shows the results of cell typing analysis that can be performed using spatial abundance data collected using the method of this disclosure. [Figure 6C] Figure 6C shows the results of differential expression analysis induced by cell interactions, which can be performed using spatial abundance data collected using the method of this disclosure. [Figure 6D] Figure 6D shows images of individual target analytes detected in melanoma FFPE tissue samples using the method of this disclosure. [Figure 6E] Figure 6E shows images of individual target analytes detected in non-small cell lung cancer (NSCLC) FFPE tissue samples using the methods of this disclosure. [Figure 6F]Figure 6F shows images of individual target analytes detected in a renal cell carcinoma FFPE tissue sample using the method of this disclosure. [Figure 6G] Figure 6G shows images of individual target analytes detected in colorectal cancer (CRC) and tonsil FFPE tissue samples using the method of this disclosure. Detailed description of the invention

[0038] This disclosure provides a method for preparing a biological sample for fluorescence imaging. The disclosure also provides in situ hybridization (ISH) probes and reporter probes for use in the method of this disclosure, as well as kits comprising these ISH probes and reporter probes. The disclosure also provides a method for determining the abundance and spatial arrangement of at least two target nucleic acid molecules in a biological sample.

[0039] Sample processing method

[0040] In some embodiments, the Disclosure provides a method comprising: a) mounting a biological sample onto a functionalized microscope slide to prepare a mounted biological sample, wherein the biological sample is a formalin-fixed paraffin-embedded (FFPE) microtome section; b) calcining the mounted biological sample; c) deparaffinizing the mounted biological sample; d) performing a targeted activation reaction on the mounted biological sample; e) permeabilizing the mounted biological sample; f) applying at least one reference marker to the mounted biological sample; g) fixing the mounted biological sample; h) contacting the mounted biological sample with at least one nucleic acid probe; and i) washing the mounted biological sample.

[0041] In some embodiments, the above method may optionally further include the step of j) dehydrating the attached biological sample.

[0042] In some embodiments, the method described above may further include assembling the fitted biological sample into a flow cell after step (i) or after step (j).

[0043] In some embodiments, the method described above may further include incubating the fitted biological sample in a blocking solution after step (g) and before step (h).

[0044] In some embodiments, the method described above may further include irradiating the biological sample with blue light and / or UV light before or after any of the steps to quench the autofluorescence of the sample via photobleaching. In some embodiments, any combination of UV irradiation and readout channel irradiation can be used to quench the autofluorescence of the sample via photobleaching. In some embodiments, irradiation can be performed simultaneously with any of the above steps, including but not limited to step (h). In some embodiments, irradiation can be performed using low-dose irradiation over a long period of time.

[0045] In some embodiments, the functionalized microscope slide may be a microscope slide functionalized with (3-aminopropyl)trimethoxysilane (APTMS). In some embodiments, an APTMS-functionalized microscope slide can be prepared by a method comprising: a) cleaning the microscope slide using a plasma instrument; b) incubating the microscope slide in a 0.5% APTMS solution for about 1 minute; c) sonicating the microscope slide in a 0.5% APTMS solution for about 10 seconds; d) repeating steps (b) and (c) twice so that the microscope slide is immersed in the 0.5% APTMS solution for about 3.5 minutes; e) rinsing the microscope slide with water at least three times; and f) drying the microscope slide under nitrogen.

[0046] In some embodiments, the functionalized microscope slide can be any positively charged microscope slide. As will be understood by those skilled in the art, non-limiting examples of commercially available positively charged microscope slides include, but are not limited to, poly-L-lysine coated glass slides, Leica BOND Plus® slides, and Fisherbrand® SuperFrost® Plus slides.

[0047] In some aspects of the method of the present disclosure, the step of mounting a biological sample onto a functionalized microscope slide may include mounting the biological sample onto the functionalized microscope slide and drying the mounted biological sample at room temperature for at least about 12 hours, or at least about 13 hours, or at least about 14 hours, or at least about 15 hours, or at least about 16 hours, or at least about 17 hours, or at least about 18 hours.

[0048] In some embodiments of the methods of the present disclosure, the step of calcining the attached biological sample may include calcining the attached biological sample at at least about 50°C, or at least about 55°C, or at least about 60°C, or at least about 65°C, or at least about 70°C, or at least about 75°C, or at least about 80°C. In some embodiments, the step of calcining the attached biological sample may include calcining the attached biological sample at about 60°C.

[0049] In some embodiments of the method of this disclosure, the step of calcining the attached biological sample may include calcining the attached biological sample for at least about 0.5 hours, or at least about 1 hour, or at least about 1.5 hours, or at least about 2 hours. In some embodiments, the step of calcining the attached biological sample may include calcining the attached biological sample for about 1 hour.

[0050] In some aspects of the method disclosed herein, the step of calcining the attached biological sample may include calcining the attached biological sample at approximately 60°C for approximately 1 hour.

[0051] In some embodiments of the method of this disclosure, the step of deparaffinizing the attached biological sample includes: a) incubating the attached biological sample in a first xylene solution for about 5 minutes; b) incubating the attached biological sample in a second xylene solution for about 5 minutes; c) incubating the attached biological sample in a first 100% ethanol solution for about 2 minutes; d) incubating the attached biological sample in a second 100% ethanol solution for about 2 minutes; and e) drying the attached biological sample at about 60°C for about 5 minutes. In some embodiments, incubation in the first xylene solution and / or the second xylene solution may include stirring the attached biological sample in the xylene solution, for example by shaking the biological sample up and down in the solution.

[0052] While not bound by theory, FFPE samples contain DNA molecules that are cross-linked to each other, as well as DNA molecules that are cross-linked to RNA and protein molecules. Therefore, disrupting these cross-links can facilitate the release of DNA for subsequent purification. Disruption of these cross-links can be achieved by performing a targeted retrieval (or antigen retrieval) reaction on a biological sample such as an FFPE sample. In a targeted retrieval reaction, a biological sample such as an FFPE sample can be incubated with a targeted retrieval solution, which is suitable for removing cross-links between DNA, RNA, and proteins in the biological sample, thereby enabling the recovery of analyzable biomolecules.

[0053] In some embodiments, the target activating solution may have a pH of about 8.0 to about 10.0. In some embodiments, the target activating solution may have a pH of about 8.5 to about 9.5. In some embodiments, the target activating solution may have a pH of about 9.0. In some embodiments, the target activating solution may contain a buffer. In some embodiments, the buffer may be Tris (TRIS).

[0054] In some embodiments, the target activating solution may contain a chelating agent. In some embodiments, the chelating agent may be ethylenediaminetetraacetic acid (EDTA). In some embodiments, the target activating solution may contain about 0.1 to about 2 mM of EDTA. In some embodiments, the target activating solution may contain about 0.5 to about 1.5 mM of EDTA. In some embodiments, the target activating solution may contain about 1.0 mM of EDTA.

[0055] In some embodiments, the target retrieval solution may be a solution of Tris and EDTA. In some embodiments, the target retrieval solution may be a solution of approximately 10 mM TRIS and approximately 1 mM EDTA at pH 9.0.

[0056] In some embodiments, the target retrieval solution may be RNAscope® Target Retrieval Solution (ACD).

[0057] In some embodiments of the methods of this disclosure, the step of performing a targeted retrieval reaction on an attached biological sample may include incubating the attached biological sample in a targeted retrieval solution at approximately 100°C, where the attached biological sample is incubated in the targeted retrieval solution at approximately 100°C for a time specific to the type of attached biological sample. In a non-limiting example where the attached biological sample is a human breast cancer sample, the attached biological sample may be incubated in the targeted retrieval solution at approximately 100°C for approximately 15 minutes. Incubation times for various sample types are shown in Table 1. In some embodiments, the step of performing a targeted retrieval reaction may further include incubating the attached biological sample in water for at least approximately 15 seconds after incubation in the targeted retrieval solution; incubating the attached biological sample in a solution of 100% ethanol for at least approximately 3 minutes; and drying the attached biological sample. In some embodiments, the water may be diethyl pyrocarbonate (DEPC) treated water.

[0058] Therefore, the step of performing a targeted activation reaction on the attached biological sample may include a) incubating the attached biological sample in a targeted activation solution at approximately 100°C for the time specified in Table 1, b) incubating the attached biological sample in DEPC-treated water for approximately 15 seconds, c) incubating the attached biological sample in a 100% ethanol solution for approximately 3 minutes, and d) drying the attached biological sample. [Table 1]

[0059] In some aspects of the method disclosed herein, the step of permeabilizing the attached biological sample may include incubating the attached biological sample in a proteinase K solution.

[0060] In some embodiments, the proteinase K solution has a proteinase K concentration of at least about 0.1 μg / mL, or at least about 0.25 μg / mL, or at least about 0.5 μg / mL, or at least about 0.75 μg / mL, or at least 1 μg / mL, or at least about 1.25 μg / mL, or at least about 1.5 μg / mL, or at least about 1.75 μg / mL, or at least about 2 μg / mL, or at least about 2.25 μg The solution is at a concentration of 1 μg / mL, or at least about 2.5 μg / mL, or at least about 2.75 μg / mL, or at least about 3 μg / mL, or at least about 3.25 μg / mL, or at least about 3.5 μg / mL, or at least about 3.75 μg / mL, or at least about 4 μg / mL, or at least about 4.25 μg / mL, or at least about 4.5 μg / mL, or at least about 4.75 μg / mL, or at least about 5 μg / mL. In some embodiments, the proteinase K solution is a solution in which the concentration of proteinase K is about 1 μg / mL. In some embodiments, the proteinase K solution is a solution in which proteinase K is diluted in phosphate-buffered saline (PBS). In some embodiments, the proteinase K solution is a solution in which proteinase K is diluted in a protease cocktail, including but not limited to ACD Protease Plus®.

[0061] In some embodiments, PBS may contain a combination of NaCl, KCl, Na2HPO4, and KH2PO4. In some embodiments, PBS may contain a solution of 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4 at pH 7.4. Thus, in some embodiments, the proteinase K solution is a solution in which the concentration of proteinase K is approximately 1 μg / mL in PBS, where PBS contains 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4 at pH 7.4.

[0062] In some embodiments, the step of permeabilizing the attached biological sample may include incubating the attached biological sample in a proteinase K solution at approximately 40°C. The step of permeabilizing the attached biological sample may include incubating the attached biological sample in a proteinase K solution at approximately 40°C for a time specific to the type of attached biological sample. In the non-limiting example where the attached biological sample is a human breast cancer sample, the attached biological sample may be incubated in a proteinase K solution at approximately 40°C for approximately 30 minutes. Table 2 shows incubation times for various sample types.

[0063] In some embodiments, the permeabilization step of the attached biological sample may include incubation of the attached biological sample in a proteinase solution at approximately 40°C. In some embodiments, permeabilization of the attached biological sample may include incubation of the attached biological sample in a proteinase solution at approximately 40°C for a duration specific to the type of attached biological sample. In the non-limiting example where the attached biological sample is a human breast cancer sample, the attached biological sample may be incubated in a proteinase solution at approximately 40°C for approximately 30 minutes. Table 2 shows incubation times for various sample types.

[0064] In some embodiments, the proteinase solution may contain a solution of protease K at a concentration of about 0.1 to about 5.0 μg / mL, or a solution of protease K at a concentration of 0.1 to 5.0 μg / mL. In some embodiments, the proteinase solution may contain a solution of protease K at a concentration of about 0.1 to about 5.0 μg / mL, or a solution of protease K at a concentration of 0.1 to 5.0 μg / mL in PBS. In some embodiments, the proteinase solution may contain a protease K solution at a concentration of about 0.1 to about 5.0 μg / mL, or 0.1 to 5.0 μg / mL, in a protease cocktail (e.g., Protease Plus solution from ACD). In some embodiments, the proteinase solution may contain a protease cocktail known in the art, for example, Protease Plus solution from ACD. [Table 2]

[0065] In some embodiments, incubating the attached biological sample in a proteinase K solution may further include, for example, drawing a hydrophobic barrier around the attached biological sample with a PAP pen.

[0066] In some embodiments, the step of permeabilizing the attached biological sample may include incubating the biological sample in a proteinase K solution for at least about 30 minutes in a container lined with paper (e.g., Kimwipes or a suitable substitute) moistened with DEPC-treated water and preheated to about 40°C.

[0067] In some embodiments, the step of permeabilizing the attached biological sample may further include incubating the attached biological sample in a proteinase K solution, followed by washing the attached biological sample with water. The water may be DEPC-treated water. In some embodiments, washing the attached biological sample with water may include washing the attached biological sample with a first aliquot of DEPC-treated water, and then washing the attached biological sample with a second aliquot of DEPC-treated water.

[0068] Therefore, the step of permeabilizing the attached biological sample may include a) incubating the attached biological sample in a proteinase K solution at approximately 40°C for the time specified in Table 2, where the concentration of proteinase K in the proteinase K solution is approximately 1 μg / mL, b) washing the biological sample with a first aliquot of DEPC-treated water, and c) washing the biological sample with a second aliquot of DEPC-treated water.

[0069] Therefore, the process of permeabilizing the attached biological sample may include a) incubating the attached biological sample in a proteinase solution at approximately 40°C for the time specified in Table 2, wherein the proteinase solution contains a concentration of approximately 0.1 to approximately 5.0 μg / mL, or a proteinase K solution containing a concentration of 0.1 to 5.0 μg / mL; b) washing the biological sample with a first aliquot of DEPC-treated water; and c) washing the biological sample with a second aliquot of DEPC-treated water.

[0070] In some embodiments of the methods of the present disclosure, the step of applying at least one reference marker to an attached biological sample may include incubating the attached biological sample in a solution containing at least one reference marker. As will be understood by those skilled in the art, the at least one reference marker may be any reference marker known in the art to be useful for fluorescence imaging. In some embodiments, the at least one reference marker may be diluted in a 2× saline-sodium citrate (SSC) solution. In some embodiments, the at least one reference marker may be diluted in a 2× saline-sodium citrate-Tween® (SSCG) solution. In some embodiments, the attached biological sample may be incubated in a solution containing at least one reference marker for at least about 1 minute, or at least about 2 minutes, or at least about 3 minutes, or at least about 4 minutes, or at least about 5 minutes, or at least about 6 minutes, or at least about 7 minutes, or at least about 8 minutes, or at least about 9 minutes, or at least about 10 minutes. In some embodiments, the attached biological sample may be incubated in a solution containing at least one reference marker for about 5 minutes. In some embodiments, the attached biological sample can be incubated in a solution containing at least one reference marker at approximately room temperature. In some embodiments, after incubation with the solution containing at least one reference marker, the attached biological sample can be washed with, for example, phosphate buffer solution (PBS). In some embodiments, before applying the solution containing at least one reference marker to the attached biological sample, the solution can be stirred (e.g., vortex stirred) for at least 30 seconds.

[0071] In some aspects of the methods of the present disclosure, the 2×SSC buffer may contain about 300 mM NaCl and about 30 mM sodium citrate. In some aspects of the methods of the present disclosure, the 2×SSC buffer may contain 300 mM NaCl and 30 mM sodium citrate.

[0072] In some aspects of the methods of the present disclosure, the 2×SSCT buffer may contain about 0.1% Tween20, about 300 mM NaCl, and about 30 mM sodium citrate.

[0073] In some embodiments, at least one reference marker can be a 200 nm carboxylated microsphere stained red, blue, yellow, and / or green. In some embodiments, a solution containing at least one reference marker can contain 200 nm carboxylated microspheres stained red, blue, and / or green at concentrations of at least about 0.00025%, or at least about 0.0005%, or at least about 0.00075%, or at least about 0.001%, or at least about 0.00125%, or at least about 0.0015%, or at least about 0.00175%, or at least about 0.002%, or at least about 0.005%, or at least about 0.01%. In some embodiments, a solution containing at least one reference marker can contain 200 nm carboxylated microspheres stained red, blue, and / or green at a concentration of about 0.001%. In some embodiments, at least one reference marker may be carboxylated microspheres stained red, yellow, blue, and / or green (e.g., carboxylated microspheres of 200 nm). In some embodiments, a solution containing at least one reference marker may contain carboxylated microspheres stained red, yellow, blue, and / or green at concentrations of about 0.00025%, or at least about 0.0005%, or at least about 0.00075%, or at least about 0.001%, or at least about 0.00125%, or at least about 0.0015%, or at least about 0.00175%, or at least about 0.002%, or at least about 0.005%, or at least about 0.01%. In some embodiments, a solution containing at least one reference marker may contain carboxylated microspheres stained red, yellow, blue, and / or green at concentrations of about 0.001%. In some embodiments, a solution containing at least one reference marker may contain carboxylated microspheres stained red, yellow, blue, and / or green at concentrations of about 0.0005% to about 0.003%, or 0.0005% to 0.003%.

[0074] In some embodiments, at least one reference marker may be fluorescent nanodiamond (FND). In some embodiments, the FND may be non-carboxylated FND. In some embodiments, a solution containing at least one reference marker may contain FND at a concentration of at least about 0.0001%, or at least about 0.00015%, or at least about 0.0002%, or at least about 0.00025%, or at least about 0.0003%, or at least about 0.00035%, or at least about 0.0004%, or at least about 0.00045%, or at least about 0.0005%, or at least about 0.00055%, or at least about 0.001%. In some embodiments, a solution containing at least one reference marker may contain FND at a concentration of about 0.00045%.

[0075] In some embodiments, a solution containing at least one reference marker may contain a combination of at least two reference markers. In non-limiting examples, a solution containing at least one reference marker may contain red, blue, and / or green 200 nm carboxylated microspheres and non-carboxylated FNDs. In non-limiting examples, a solution containing at least one reference marker may contain red, blue, and / or green 200 nm carboxylated microspheres at a concentration of about 0.001% and non-carboxylated FNDs at a concentration of about 0.00045%.

[0076] In some embodiments, a solution containing at least one reference marker may contain a combination of at least two reference markers. In non-limiting examples, a solution containing at least one reference marker may contain carboxylated microspheres stained red, yellow, blue, and / or green, and non-carboxylated FNDs. In non-limiting examples, a solution containing at least one reference marker may contain nm carboxylated microspheres stained red, blue, and / or green at concentrations of about 0.0005% to about 0.003%, or 0.0005% to 0.003%, and non-carboxylated FNDs at a concentration of about 0.00045%.

[0077] In some embodiments, a solution containing at least one reference marker can be prepared by diluting at least one reference marker in a suitable buffer containing but not limited to a 2×SSC solution, then stirring the solution for about 1 minute (e.g., vortex stirring), then sonicating the solution for about 2 minutes, then stirring the solution again for about 1 minute, and then sonicating the solution again for about 2 minutes.

[0078] In some embodiments, a solution containing at least one reference marker can be prepared by diluting at least one reference marker in a suitable buffer containing but not limited to a 2×SSCT solution, stirring the solution for about 1 minute (e.g., vortex stirring), sonicating for about 1 minute, stirring again for about 1 minute, and then sonicating again for about 2 minutes.

[0079] Therefore, the step of applying at least one reference marker to an attached biological sample may include a) incubating the attached biological sample in a solution containing at least one reference marker at room temperature for about 5 minutes, where the solution containing at least one reference marker is a solution containing about 0.001% red, blue and / or green carboxylated microspheres and about 0.00045% uncarboxylated FND in a 2×SSC solution, and b) washing the attached biological sample with 1×PBS.

[0080] Therefore, the step of applying at least one reference marker to an attached biological sample may include a) incubating the attached biological sample in a solution containing at least one reference marker for about 5 minutes at about room temperature, where the solution containing at least one reference marker is a solution containing carboxylated microspheres stained red, yellow, blue and / or green at a concentration of about 0.0005% to about 0.003%, or 0.0005% to 0.003%, and b) washing the attached biological sample with 1×PBS.

[0081] In some embodiments of the methods of the present disclosure, the step of fixing the attached biological sample may include incubating the attached biological sample in a neutral buffered formalin (NBF) solution, then incubating the attached biological sample in Tris-glycine buffer, and then incubating the attached biological sample in 1×PBS. In some embodiments, the concentration of NBF in the NBF solution may be at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%. In some embodiments, the concentration of NBF in the NBF solution may be about 10%. In some embodiments, each incubation step in fixing the attached biological sample may last at least about 1 minute, or at least about 2 minutes, or at least about 3 minutes, or at least about 4 minutes, or at least about 5 minutes, or at least about 6 minutes, or at least about 7 minutes, or at least about 8 minutes, or at least about 9 minutes, or at least about 10 minutes. In some embodiments, each incubation step in fixing the mounted biological sample may be approximately 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes. In some embodiments, each incubation step may be approximately 5 minutes. In some embodiments, each incubation step may be approximately 1 minute. In some embodiments, incubation of the mounted biological sample in Tris-glycine buffer may include incubation of the mounted biological sample in a first Tris-glycine buffer, followed by incubation of the mounted biological sample in a second Tris-glycine buffer.

[0082] The process of fixing the mounted biological sample may include a) incubating the mounted biological sample in 10% NBF for about 5 minutes; b) incubating the mounted biological sample in a first Tris-glycine buffer for about 5 minutes; c) incubating the mounted biological sample in a second Tris-glycine buffer for about 5 minutes; and d) incubating the mounted biological sample in 1×PBS for about 5 minutes.

[0083] Therefore, the steps for fixing the mounted biological sample may include a) incubating the mounted biological sample in 10% NBF for about 1 minute; b) incubating the mounted biological sample in a first Tris-glycine buffer for about 5 minutes; c) incubating the mounted biological sample in a second Tris-glycine buffer for about 5 minutes; and d) incubating the mounted biological sample in 1×PBS for about 5 minutes.

[0084] In some embodiments of the methods of this disclosure, the step of incubating the attached biological sample in a blocking solution may include incubating the attached biological sample in a sulfo-NHS-acetic acid / Tween® 20 solution. In some embodiments, the sulfo-NHS-acetic acid / Tween 20 solution may contain about 100 mM sulfo-NHS-acetic acid and about 0.5% Tween 20 in about 100 mM sodium phosphate pH 8. In some embodiments, the sulfo-NHS-acetic acid / Tween 20 solution may contain 100 mM sulfo-NHS-acetic acid and 0.5% Tween 20 in about 100 mM sodium phosphate pH 8. In some embodiments, the attached biological sample may be incubated in the sulfo-NHS-acetic acid / Tween 20 solution for at least about 5 minutes, or at least about 10 minutes, or at least about 15 minutes, or at least about 20 minutes. In some embodiments, the attached biological sample can be incubated in a sulfo-NHS-acetic acid / Tween20 solution for about 5 minutes, or about 10 minutes, or about 15 minutes, or about 20 minutes. In some embodiments, the attached biological sample can be incubated in a sulfo-NHS-acetic acid / Tween20 solution for about 15 minutes.

[0085] In some aspects of the methods of the present disclosure, the step of incubating the attached biological sample in a blocking solution may include incubating the sample in sulfo-NHS-acetic acid / Tween20, and then incubating the attached biological sample in 1×PBS for at least about 1 minute, or at least about 2 minutes, or at least about 3 minutes, or at least about 4 minutes, or at least about 5 minutes, or at least about 6 minutes, or at least about 7 minutes, or at least about 8 minutes, or at least about 9 minutes, or at least about 10 minutes. In some aspects of the methods of the present disclosure, the step of incubating the attached biological sample in a blocking solution may include incubating the attached biological sample in a sulfo-NHS-acetic acid / Tween20 solution, and then incubating the attached biological sample in 1×PBS for at least 1 minute, or at least about 2 minutes, or at least 3 minutes, or at least 4 minutes, or at least 5 minutes, or at least 6 minutes, or at least 7 minutes, or at least 8 minutes, or at least 9 minutes, or at least 10 minutes. In some aspects of the method of the present disclosure, the step of incubating the attached biological sample in a blocking solution may include incubating the attached biological sample in a sulfo-NHS-acetic acid / Tween20 solution, and then incubating the attached biological sample in 1×PBS for about 5 minutes.

[0086] Therefore, the step of incubating the attached biological sample in a blocking solution may include: i) incubating the attached biological sample in a sulfo-NHS-acetic acid / Tween20 solution for about 15 minutes, where the sulfo-NHS-acetic acid / Tween20 solution comprises about 100 mM sulfo-NHS-acetic acid and about 0.5% Tween20 in about 100 mM sodium phosphate pH 8; and ii) incubating the attached biological sample in 1×PBS for about 5 minutes.

[0087] In some embodiments of the methods of the present disclosure, contacting an attached biological sample with at least one nucleic acid probe may include incubating the attached biological sample with a solution containing multiple ISH probes of the present disclosure. In some embodiments, the attached biological sample may be incubated with a solution containing multiple ISH probes for at least about 12 hours, or at least about 13 hours, or at least about 14 hours, or at least about 15 hours, or at least about 16 hours, or at least about 17 hours, or at least about 18 hours, or at least about 19 hours, or at least about 20 hours, or at least about 21 hours, or at least about 22 hours, or at least about 23 hours, or at least about 24 hours. In some embodiments, the attached biological sample may be incubated with a solution containing multiple ISH probes for about 16 to about 18 hours.

[0088] In some embodiments, the attached biological sample can be incubated with a solution containing multiple ISH probes at a temperature of at least about 35°C, or at least about 36°C, or at least about 37°C, or at least about 38°C, or at least about 39°C, or at least about 40°C. In some embodiments, the attached biological sample can be incubated with a solution containing multiple ISH probes at a temperature of about 35°C.

[0089] In some embodiments, a solution containing multiple ISH probes of the Disclosure may contain a single type of ISH probe. In some embodiments, a solution containing multiple ISH probes of the Disclosure may contain at least about 2, or at least about 3, or at least about 4, or at least about 5, or at least about 6, or at least about 7, or at least about 8, or at least about 9, or at least about 10, or at least about 25, or at least about 50, or at least about 75, or at least about 100, or at least about 250, or at least about 500, or at least about 750, or at least about 1,000, or at least about 5,000, or at least about 10,000, or at least about 15,000, or at least about 20,000, or at least about 50,000, or at least about 100,000, or at least about 500,000, or at least about 1,000,000 different types of ISH probes.

[0090] In some embodiments, the concentration of at least one ISH probe among a plurality of ISH probes may be at least about 0.01 nM, or at least about 0.1 nM, or at least about 1 nM, or at least about 5 nM, or at least about 10 nM, or at least about 25 nM, or at least about 50 nM, or at least about 75 nM, or at least about 100 nM, or at least about 125 nM, or at least about 150 nM, or at least about 175 nM, or at least about 200 nM, or at least about 300 nM, or at least about 400 nM, or at least about 500 nM. In some embodiments, the concentration of at least one ISH probe among multiple ISH probes may be about 0.01 nM, or about 0.1 nM, or about 1 nM, or about 5 nM, or about 10 nM, or about 25 nM, or about 50 nM, or about 75 nM, or about 100 nM, or about 125 nM, or about 150 nM, or about 175 nM, or about 200 nM, or about 300 nM, or about 400 nM, or about 500 nM. In some embodiments, the concentration of at least one ISH probe among multiple ISH probes may be about 200 nM. In some embodiments, the concentration of at least one ISH probe among multiple ISH probes may be about 1 nM.

[0091] In some embodiments, the various ISH probe concentrations of multiple ISH probes may be at least about 0.01 nM, or at least about 0.1 nM, or at least about 1 nM, or at least about 5 nM, or at least about 10 nM, or at least about 25 nM, or at least about 50 nM, or at least about 75 nM, or at least about 100 nM, or at least about 125 nM, or at least about 150 nM, or at least about 175 nM, or at least about 200 nM, or at least about 300 nM, or at least about 400 nM, or at least about 500 nM. In some embodiments, the various ISH probe concentrations of multiple ISH probes may be approximately 0.01 nM, or approximately 0.1 nM, or approximately 1 nM, or approximately 5 nM, or approximately 10 nM, or approximately 25 nM, or approximately 50 nM, or approximately 75 nM, or approximately 100 nM, or approximately 125 nM, or approximately 150 nM, or approximately 175 nM, or approximately 200 nM, or approximately 300 nM, or approximately 400 nM, or approximately 500 nM. In some embodiments, the various concentrations of multiple ISH probes may be approximately 200 nM. In some embodiments, the various concentrations of multiple ISH probes may be approximately 1 nM.

[0092] In some embodiments, a solution containing multiple ISH probes may include at least one ISH probe that includes a target-binding domain designed not to specifically bind to any target analyte in a biological sample (e.g., a target nucleic acid molecule and / or a target protein molecule). In some embodiments, a solution containing multiple ISH probes may include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 50, at least 100, or at least 1000 ISH probes that include a target-binding domain designed not to specifically bind to any target analyte in a biological sample (e.g., a target nucleic acid molecule and / or a target protein molecule). These ISH probes that include a target-binding domain designed not to specifically bind to any target analyte are referred to herein as “negative ISH probes”. A non-limiting example of a negative ISH probe is an ISH probe containing a target-binding domain that is a single-stranded nucleic acid, where the sequence of the single-stranded nucleic acid is designed not to be complementary to any known sequence specific to the biological sample being analyzed and / or any known sequence existing on Earth. As will be understood by those skilled in the art, examples of such sequences include those published by the External RNA Control Consosium (ERCC) evaluation. While we do not wish to be bound by theory, the use of these negative ISH probes in the methods of this disclosure makes it possible for those skilled in the art to determine the level of background noise in the results from the biological sample. As will be understood by those skilled in the art, since a negative ISH probe should not bind to any target analyte, any signal originating from the negative ISH probe that is recorded represents non-specific binding of the ISH probe to the sample (i.e., background noise). In some embodiments, those skilled in the art can use the level of background noise detected by the negative ISH probe to more accurately determine the absolute abundance of the target analyte in the biological sample.

[0093] In some embodiments, a solution containing multiple ISH probes may include ISH probes diluted with buffer R (Retriever buffer).

[0094] In some embodiments, buffer R may contain at least one of dextran sulfate, bovine serum albumin (BSA), single-stranded DNA (ssDNA), physiological saline-sodium citrate (SSC), and formamide. In some embodiments, buffer R may contain a combination of dextran sulfate, BSA, ssDNA, SSC, and formamide. In some embodiments, the single-stranded DNA may contain salmon sperm DNA.

[0095] In some embodiments, the ISH probe can be diluted with buffer R so that the final concentration of dextran sulfate is approximately 0.5% to approximately 4.5%, or approximately 1.5% to approximately 3.5%. In some embodiments, the ISH probe can be diluted with buffer R so that the final concentration of dextran sulfate is approximately 2.5%.

[0096] In some embodiments, the ISH probe can be diluted with buffer R so that the final concentration of BSA is approximately 0.01% to approximately 2%, or approximately 0.1% to approximately 1%, and in some embodiments, the ISH probe can be diluted with buffer R so that the final concentration of BSA is approximately 0.2%.

[0097] In some embodiments, the ISH probe can be diluted in buffer R to a final ssDNA concentration of approximately 0.01 mg / ml to approximately 1 mg / ml, or approximately 0.05 mg / ml to approximately 0.5 mg / ml. In some embodiments, the ISH probe can be diluted in buffer R to a final ssDNA concentration of approximately 0.1 mg / ml.

[0098] In some embodiments, the ISH probe can be diluted with buffer R so that the final concentration of SSC is approximately 0.5 × (times) to approximately 3.5 × or approximately 1 × to approximately 3 ×. In some embodiments, the ISH probe can be diluted with buffer R so that the final concentration of SSC is approximately 2 ×.

[0099] In some embodiments, the ISH probe can be diluted with buffer R so that the final concentration of formamide is approximately 20% to approximately 60%, or approximately 30% to approximately 50%. In some embodiments, the ISH probe can be diluted with buffer R so that the final concentration of formamide is approximately 40%.

[0100] In some embodiments, the ISH probe can be diluted with buffer R such that the final concentration of dextran sulfate is approximately 2.5%, the final concentration of BSA is approximately 0.2%, the final concentration of ssDNA is approximately 0.1 mg / mL, the final concentration of SSC is approximately 2×, and the final concentration of formamide is approximately 40%.

[0101] In some embodiments, the solution containing multiple ISH probes may further contain, but are not limited to, RNase inhibitors, including SUPERase-In® RNase inhibitors. The concentration of RNase may be approximately 0.1 units / μL.

[0102] In some embodiments, the ISH probes can be initially denatured by first incubating the ISH probes at approximately 95°C for about 2 minutes, and then immediately cooling the ISH probes on ice for about 1 minute, before incubating the attached biological sample with a solution containing multiple ISH probes.

[0103] In some embodiments, the attached biological sample can be rinsed with an RNase inhibitor solution and incubated with a solution containing multiple ISH probes in a container lined with paper (e.g., Kimwipes or a suitable alternative) moistened with DEPC-treated water.

[0104] Therefore, the step of bringing the attached biological sample into contact with at least one nucleic acid probe includes a) incubating the attached biological sample with a solution containing a plurality of ISH probes of the present disclosure at approximately 37°C for approximately 16 to approximately 18 hours, wherein the solution contains at least one ISH probe, and at least one of the plurality of ISH probes is present at a concentration of approximately 200 nM.

[0105] In some aspects of the method of the present disclosure, the step of washing the attached biological sample may include a) incubating the attached biological sample in a first 2×SSC solution, b) incubating the attached biological sample in a first formamide solution, c) incubating the attached biological sample in a second formamide solution, d) incubating the attached biological sample with a second 2×SSC solution, and e) incubating the attached biological sample with a third 2×SSC solution.

[0106] In some embodiments, the formamide solution may be formamide in a 2×SSC solution. In some embodiments, the concentration of formamide may be at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%. In some embodiments, the concentration of formamide may be about 50%. In some embodiments, the attached biological sample may be incubated with the first formamide solution and / or the second formamide solution for at least about 15 minutes, or at least about 20 minutes, or at least about 25 minutes, or at least about 30 minutes, or at least about 35 minutes, or at least about 40 minutes. In some embodiments, the attached biological sample may be incubated with the first formamide solution and / or the second formamide solution for about 25 minutes.

[0107] In some embodiments, the attached biological sample can be incubated with a second 2×SSC solution and / or a third 2×SSC solution for at least about 0.5 minutes, or at least about 1 minute, or at least about 1.5 minutes, or at least about 2.0 minutes, or at least about 2.5 minutes, or at least 3.0 minutes, or at least about 3.5 minutes, or at least about 4.0 minutes, or at least about 4.5 minutes, or at least about 5 minutes. In some embodiments, the attached biological sample can be incubated with a second 2×SSC solution and / or a third 2×SSC solution for about 2 minutes.

[0108] Therefore, the step of washing the attached biological sample may include a) incubating the attached biological sample with a first 2×SSC solution, b) incubating the attached biological sample in a first 50% formamide in a 2×SSC solution for about 25 minutes, c) incubating the attached biological sample with a second 50% formamide in a 2×SSC solution for about 25 minutes, d) incubating the attached biological sample with a second 2×SSC solution for about 2 minutes, and e) incubating the attached biological sample with a third 2×SSC solution for about 2 minutes.

[0109] In some embodiments of the methods of the present disclosure, the step of dehydrating the attached biological sample may include incubating the attached biological sample under an ethanol gradient, as can be understood by those skilled in the art. In some embodiments, incubating the attached biological sample under an ethanol gradient may include a) incubating the attached biological sample in a 70% ethanol solution for about 3 minutes, b) incubating the attached biological sample in an 85% ethanol solution for about 3 minutes, and c) incubating the attached biological sample in a 100% ethanol solution for about 3 minutes.

[0110] In some embodiments, the biological sample may be an FFPE microtome section having a thickness of at least about 1 μm, or at least about 2 μm, or at least about 3 μm, or at least about 4 μm, or at least about 5 μm, or at least about 6 μm, or at least about 7 μm, or at least about 8 μm, or at least about 9 μm, or at least about 10 μm. In some embodiments, the biological sample may be an FFPE microtome section having a thickness of about 5 μm.

[0111] In some embodiments, the biological sample may be a tissue sample from any organ. In some embodiments, the biological sample may be a tissue sample from the intestines, embryo, brain, spleen, eye, retina, liver, kidney, breast, throat, colon, lung, prostate, lymph nodes, tonsils, pancreas, cervix, head, neck, liver, skin, nerves, placenta, or any other organ.

[0112] In some embodiments, the biological sample may contain non-cancerous cells. In some embodiments, the biological sample may contain cancer cells. In some embodiments, the biological sample may contain both non-cancerous and cancer cells. Cancer cells may be carcinomas, lymphomas, blastomas, sarcomas, leukemias, and germ cell tumors. Cancer cells can originate from adrenocortical carcinoma, urothelial carcinoma of the bladder, invasive breast carcinoma, cervical squamous cell carcinoma, cervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, lymphocyte-mediated diffuse large B-cell lymphoma, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe, renal pellucid cell carcinoma, renal papillary cell carcinoma, acute myeloid leukemia, cerebral glioma, hepatocellular carcinoma of the liver, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, ovarian serous cyst adenocarcinoma, pancreatic adenocarcinoma, pheochromocytoma, paraganglioma, prostate adenocarcinoma, rectal adenocarcinoma, sarcoma, cutaneous melanoma, gastric adenocarcinoma, testicular germ cell tumor, thyroid cancer, thymic carcinoma, uterine carcinosarcoma, or uveal melanoma. Other examples include breast cancer, lung cancer, lymphoma, melanoma, liver cancer, colorectal cancer, ovarian cancer, bladder cancer, kidney cancer, or gastric cancer.Further examples of cancer include neuroendocrine cancer, non-small cell lung cancer (NSCLC), small cell lung cancer, thyroid cancer, endometrial cancer, biliary tract cancer, esophageal cancer, anal cancer, salivary gland cancer, vulvar cancer, cervical cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenal tumors, anal cancer, bile duct cancer, bladder cancer, osteosarcoma, colorectal cancer, brain tumors, breast cancer, cancer of unknown primary origin (CUP), cancer that has metastasized to the bone, and brain cancer. Metastatic cancer, cancer that has metastasized to the liver, cancer that has metastasized to the lungs, carcinoma, cervical cancer, childhood cancer, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), colorectal cancer, ear cancer, endometrial cancer, ocular cancer, follicular dendritic cell sarcoma, gallbladder cancer, stomach cancer, gastroesophageal junction cancer, germ cell tumors, gestational trophoblastic disease (GIT), hair follicle leukemia, head and neck cancer, Hodgkin lymphoma, Kaposi's sarcoma, kidney cancer This includes, but is not limited to, laryngeal cancer, leukemia, regenerative gastric fibrosis, liver cancer, lung cancer, lymphoma, malignant Schwann cell carcinoma, intermediate germ cell tumor, melanoma, male cancer, Merkel cell carcinoma, mesothelioma, molar pregnancy, oral and oropharyngeal cancer, myeloma, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine tumor, non-Hodgkin lymphoma (NHL), esophageal cancer, ovarian cancer, pancreatic cancer, penile cancer, persistent trophoblastic disease and choriocarcinoma, pheochromocytoma, prostate cancer, pseudomyxoma peritonei, retinal cancer, retinoblastoma, salivary gland cancer, secondary cancer, signet ring cell carcinoma, skin cancer, small intestine cancer, soft tissue sarcoma, gastric cancer, T-cell pediatric non-Hodgkin lymphoma (NHL), testicular cancer, thymic cancer, thyroid cancer, tongue cancer, tonsil cancer, adrenal tumors, uterine cancer, vaginal cancer, vulvar cancer, Wilms' tumor, and gynecological cancers. Examples of cancer include, but are not limited to, hematological malignancies, lymphomas, cutaneous T-cell lymphomas, peripheral T-cell lymphomas, Hodgkin lymphomas, non-Hodgkin lymphomas, multiple myeloma, chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, myelodysplastic syndromes, myelofibrosis, biliary tract cancers, hepatocellular carcinomas, colorectal cancers, breast cancers, lung cancers, non-small cell lung cancers, ovarian cancers, thyroid cancers, renal cell carcinomas, pancreatic cancers, bladder cancers, skin cancers, malignant melanomas, Merkel cell carcinomas, uveal melanomas, or glioblastoma multiforme.

[0113] Biological samples may include, but are not limited to, humans, mice, rats, dogs, cats, sheep, rabbits, cattle, goats, or any other species, and may be derived from any species.

[0114] In situ hybridization (ISH) probes of this disclosure

[0115] Target-binding domain

[0116] This disclosure provides in situ hybridization (ISH) probes for use in the methods of this disclosure.

[0117] An ISH probe may include a target-binding domain and a barcode domain. In some embodiments, the target-binding domain is ligated to the barcode domain in an actionable manner.

[0118] In some embodiments, the target-binding domain may include a protein, peptide, aptamer, or peptoid that specifically binds to a target analyte in a biological sample. In some embodiments, the protein may be an antibody or its antigen-binding fragment. In some embodiments, the protein may be a lectin protein. In some embodiments, the protein may be any sugar-binding protein known in the art.

[0119] In some embodiments, the target-binding domain may be a single-stranded polynucleotide. The target-binding domain may include a sequence complementary to the target nucleic acid of interest identified using the method of this disclosure.

[0120] In some embodiments, the target-binding domain may have a length of at least about 35 nucleotides to at least about 40 nucleotides. In some embodiments, the target-binding domain may be about 35 to about 40 nucleotides in length. In some embodiments, the target-binding domain may have a length of about 20 nucleotides, or about 21 nucleotides, or about 22 nucleotides, or about 23 nucleotides, or about 24 nucleotides, or about 25 nucleotides, or about 26 nucleotides, or about 27 nucleotides, or about 28 nucleotides, or about 29 nucleotides, or about 30 nucleotides, or about 31 nucleotides, or about 32 nucleotides, or about 33 nucleotides, or about 34 nucleotides, or about 35 nucleotides, or about 36 nucleotides, or about 37 nucleotides, or about 38 nucleotides, or about 39 nucleotides, or about 40 nucleotides, or about 41 nucleotides, or about 42 nucleotides, or about 43 nucleotides, or about 45 nucleotides.

[0121] In some embodiments, the target-binding domain includes D-DNA. In some embodiments, the target-binding domain consists of D-DNA.

[0122] In some embodiments, the target-binding domain can be about 35 to about 40 nucleotides long and contains D-DNA.

[0123] Barcode Domain

[0124] In some embodiments, the barcode domain can be a single-stranded polynucleotide.

[0125] A barcode domain may include at least one attachment region. In some embodiments, a barcode domain may include at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten attachment regions.

[0126] In some embodiments, a barcode domain may include approximately four attachment regions.

[0127] An attachment region may include at least one nucleic acid sequence that can be reversibly bound via a reporter probe of the present disclosure. In this specification, a nucleic acid sequence that can be reversibly bound via a reporter probe of the present disclosure will be referred to as an attachment sequence. Therefore, an attachment region of a barcode domain may include at least one attachment sequence. In some embodiments, the (multiple) attachment sequences within a single attachment region may be identical, and therefore the reporter probes that bind to that single attachment region will be identical. In some embodiments, the multiple attachment sequences within a single attachment region may be different, in which case the reporter probes that bind to that single attachment region will be different.

[0128] In some embodiments, a barcode domain may include multiple attachment regions, and each attachment sequence within a different attachment region may be different, in which case different reporter probes will bind to each attachment region within the barcode domain.

[0129] In some embodiments, the attached sequence is about 5 nucleotides, or about 6 nucleotides, or about 7 nucleotides, or about 8 nucleotides, or about 9 nucleotides, or about 10 nucleotides, or about 11 nucleotides, or about 12 nucleotides, or about 13 nucleotides, or about 14 nucleotides, or about 15 nucleotides, or about 16 nucleotides, or about 17 nucleotides, or about 18 nucleotides, or about 19 nucleotides, or about 20 nucleotides in length. In some embodiments, the attached sequence may be about 14 nucleotides in length.

[0130] In some embodiments, the barcode domain includes L-DNA. In some embodiments, the barcode domain consists of L-DNA.

[0131] In some embodiments, the barcode domain may contain approximately four attachment regions, each attachment region containing approximately one attachment sequence, each attachment sequence being approximately 14 nucleotides long, the barcode domain being approximately 56 nucleotides long, each nucleic acid sequence of the attachment sequences being different, and the barcode domain containing L-DNA.

[0132] Accordingly, this disclosure provides an ISH probe comprising a target-binding domain and a barcode domain, wherein the target-binding domain is a single-stranded polynucleotide comprising a nucleic acid sequence complementary to the target nucleic acid, the target-binding domain is approximately 35 to approximately 40 nucleotides long, the target-binding domain contains D-DNA, and the barcode domain is a single-stranded polynucleotide comprising approximately 4 attachment regions, where each attachment region contains approximately 1 attachment sequence, each attachment sequence is approximately 14 nucleotides long, the sequences of each attachment sequence are different, and the barcode domain contains L-DNA. A schematic diagram of this exemplary ISH probe is shown in Figure 1.

[0133] Accordingly, the present disclosure provides an ISH probe comprising a target-binding domain and a barcode domain, wherein the target-binding domain is a single-stranded polynucleotide comprising a nucleic acid sequence complementary to the target nucleic acid, the target-binding domain is approximately 35 to approximately 40 nucleotides long, the target-binding domain is composed of D-DNA, the barcode domain is a single-stranded polynucleotide comprising approximately 4 attachment regions, each attachment region comprising approximately 1 attachment sequence, each attachment sequence being approximately 14 nucleotides long, each attachment sequence having a different sequence, and the barcode domain is composed of L-DNA.

[0134] Reporter probe of this disclosure

[0135] This disclosure provides reporter probes for use in the methods of the disclosure. The reporter probes of the disclosure bind to an attachment sequence within the attachment region of the barcode domain of the ISH probe of the disclosure. The reporter probes include at least one detectable label, such as a fluorescent moiety, which makes them detectable in the methods of the disclosure.

[0136] The reporter probe may contain at least two domains, the first domain which hybridizes to the attachment sequence, and the second domain which contains at least one detectable label.

[0137] In some embodiments, the reporter probe may include at least about 10, or at least about 15, or at least about 20, or at least about 25, or at least about 30, or at least about 35, or at least about 40, or at least about 45, or at least about 50 detectable markers. In some embodiments, the reporter probe may include at least 10, or at least about 15, or at least about 20, or at least about 25, or at least about 30, or at least about 35, or at least about 40, or at least about 45, or at least about 50 detectable markers.

[0138] In some embodiments, the reporter probe can be pre-assembled before it comes into contact with the biological sample.

[0139] In some embodiments, the reporter probe may include a first nucleic acid molecule. The first nucleic acid molecule may be a single-stranded polynucleotide. In some embodiments, the first nucleic acid molecule may include L-DNA. In some embodiments, the first nucleic acid molecule may consist of L-DNA.

[0140] The first nucleic acid molecule may include at least two domains. In some embodiments, the first domain of the first nucleic acid molecule may hybridize to an attachment sequence within the attachment region of the barcode domain of the ISH probe of this disclosure. In some embodiments, the second domain of the first nucleic acid molecule may include at least one detectable label.

[0141] In some embodiments, the second domain of the first nucleic acid molecule can hybridize to at least one second nucleic acid molecule. In some embodiments, the first nucleic acid molecule can hybridize to at least about two, or at least about three, or at least about four, or at least about five, or at least about six, or at least about seven, or at least about eight, or at least about nine, or at least about ten second nucleic acid molecules. In some embodiments, the first nucleic acid molecule can hybridize to about six second nucleic acid molecules.

[0142] In some embodiments, the first nucleic acid molecule may further include a cleavable linker moiety. In some embodiments, the cleavable linker moiety is located between a first domain and a second domain, and as a result, when the cleavable linker moiety is cleaved, the first domain and the second domain are separated. In a preferred embodiment, the cleavable linker moiety is a photocleavable linker moiety.

[0143] In some embodiments, the first domain of the first nucleic acid molecule may be about 5 nucleotides, or about 6 nucleotides, or about 7 nucleotides, or about 8 nucleotides, or about 9 nucleotides, or about 10 nucleotides, or about 11 nucleotides, or about 12 nucleotides, or about 13 nucleotides, or about 14 nucleotides, or about 15 nucleotides, or about 16 nucleotides, or about 17 nucleotides, or about 18 nucleotides, or about 19 nucleotides, or about 20 nucleotides in length. In some embodiments, the first domain of the first nucleic acid molecule may be about 14 nucleotides in length.

[0144] In some embodiments, the second domain of the first nucleic acid molecule may be about 75 nucleotides, or about 76 nucleotides, or about 77 nucleotides, or about 78 nucleotides, or about 79 nucleotides, or about 80 nucleotides, or about 81 nucleotides, or about 82 nucleotides, or about 83 nucleotides, or about 84 nucleotides, or about 85 nucleotides, or about 86 nucleotides, or about 87 nucleotides, or about 88 nucleotides, or about 89 nucleotides, or about 90 nucleotides in length. In some embodiments, the second domain of the first nucleic acid molecule may be about 84 nucleotides in length.

[0145] In some embodiments, the first nucleic acid molecule is about 90 nucleotides, or about 91 nucleotides, or about 92 nucleotides, or about 93 nucleotides, or about 94 nucleotides, or about 95 nucleotides, or about 96 nucleotides, or about 97 nucleotides, or about 98 nucleotides, or about 99 nucleotides, or about 100 nucleotides, or about 101 nucleotides, or about 102 nucleotides, or about 103 nucleotides, or about 104 nucleotides, or about 105 nucleotides, or about 106 nucleotides, or about 107 nucleotides, or about 108 nucleotides, or about 109 nucleotides, or about 110 nucleotides in length. In some embodiments, the first nucleic acid molecule may be about 98 nucleotides in length.

[0146] In some embodiments, the reporter probe may contain at least one second nucleic acid molecule. In some embodiments, the reporter probe may contain at least about two, or at least about three, or at least about four, or at least about five, or at least about six, or at least about seven, or at least about eight, or at least about nine, or at least about ten second nucleic acid molecules. In some embodiments, the reporter probe may contain about six second nucleic acid molecules. The second nucleic acid molecule may be a single-stranded polynucleotide. In some embodiments, the second nucleic acid molecule may contain L-DNA. In some embodiments, the second nucleic acid molecule may consist of L-DNA.

[0147] The second nucleic acid molecule may contain at least two domains. In some embodiments, the first domain of the second nucleic acid molecule may hybridize with the first nucleic acid molecule. In some embodiments, the second domain of the second nucleic acid molecule may contain at least one detectable label.

[0148] In some embodiments, the second nucleic acid molecule may further include a cleavable linker moiety. In some embodiments, the cleavable linker moiety may be positioned between the first and second domains, so that when the cleavable linker moiety is cleaved, the first and second domains of the second nucleic acid molecule are separated. In preferred embodiments, the cleavable linker moiety is a photocleavable linker moiety.

[0149] In some embodiments, the second domain of a second nucleic acid molecule can hybridize to at least one third nucleic acid molecule. In some embodiments, the second domain of a second nucleic acid molecule can hybridize to at least about two, or at least about three, or at least about four, or at least about five, or at least about six, or at least about seven, or at least about eight, or at least about nine, or at least about ten third nucleic acid molecules. In some embodiments, the second domain of a second nucleic acid molecule can hybridize to about five third nucleic acid molecules.

[0150] In some embodiments, the first domain of the second nucleic acid molecule may be about 5 nucleotides, or about 6 nucleotides, or about 7 nucleotides, or about 8 nucleotides, or about 9 nucleotides, or about 10 nucleotides, or about 11 nucleotides, or about 12 nucleotides, or about 13 nucleotides, or about 14 nucleotides, or about 15 nucleotides, or about 16 nucleotides, or about 17 nucleotides, or about 18 nucleotides, or about 19 nucleotides, or about 20 nucleotides in length. In some embodiments, the first domain of the second nucleic acid molecule may be about 14 nucleotides in length.

[0151] In some embodiments, the second domain of the second nucleic acid molecule may be about 65 nucleotides, or about 66 nucleotides, or about 67 nucleotides, or about 68 nucleotides, or about 69 nucleotides, or about 70 nucleotides, or about 71 nucleotides, or about 72 nucleotides, or about 73 nucleotides, or about 74 nucleotides, or about 75 nucleotides, or about 76 nucleotides, or about 77 nucleotides, or about 78 nucleotides, or about 79 nucleotides, or about 80 nucleotides, or about 81 nucleotides, or about 82 nucleotides, or about 83 nucleotides, or about 84 nucleotides, or about 85 nucleotides in length. In some embodiments, the second domain of the second nucleic acid molecule may be about 75 nucleotides in length.

[0152] In some embodiments, the reporter probe contains at least one third nucleic acid molecule. In some embodiments, the reporter probe contains at least about 20, or at least about 21, or at least about 22, or at least about 23, or at least about 24, or at least about 25, or at least about 26, or at least about 27, or at least about 28, or at least about 29, or at least about 30, at least about 31, at least about 32, at least about 33, or at least about 34, or at least about 35, or at least about 36, or at least about 37, or at least about 38, or at least about 39, or at least about 40 third nucleic acid molecules. In some embodiments, the reporter probe contains about 30 third nucleic acid molecules.

[0153] In some embodiments, the third nucleic acid molecule may include a domain that hybridizes with the second nucleic acid molecule.

[0154] In some embodiments, the third nucleic acid molecule may include at least one detectable label.

[0155] In some embodiments, the third nucleic acid molecule is about 5 nucleotides, or about 6 nucleotides, or about 7 nucleotides, or about 8 nucleotides, or about 9 nucleotides, or about 10 nucleotides, or about 11 nucleotides, or about 12 nucleotides, or about 13 nucleotides, or about 14 nucleotides, or about 15 nucleotides, or about 16 nucleotides, or about 17 nucleotides, or about 18 nucleotides, or about 19 nucleotides, or about 20 nucleotides, or about 21 nucleotides, or about 22 nucleotides, or about 23 nucleotides, or about 24 nucleotides, or about 25 nucleotides in length. In some embodiments, the third nucleic acid molecule may be about 15 nucleotides in length.

[0156] In some embodiments where the reporter probe includes multiple (two or more) detectable labels, all of the detectable labels on the reporter probe may have the same emission spectrum. In embodiments where the detectable labels are fluorescent labels, a reporter probe in which all of the detectable labels have the same emission spectrum may be referred to as a "monochromatic" reporter probe.

[0157] In some embodiments where the reporter probe includes multiple (two or more) detectable labels, the reporter probe may have two or more detectable labels, each having a different emission spectrum. In embodiments where the detectable labels are fluorescent labels, a reporter probe having two or more detectable labels, each having a different emission spectrum, may be referred to as a "multicolor" reporter probe.

[0158] This disclosure provides a reporter probe comprising a first nucleic acid molecule comprising a first domain, a second domain, and a photocleavable linker positioned between the first and second domains, wherein the second domain of the first nucleic acid molecule hybridizes to about six second nucleic acid molecules, each of which comprises a first domain, a second domain, and a photocleavable linker positioned between the first and second domains, the first domain of each of the second nucleic acid molecules hybridizes to the second domain of the first nucleic acid molecule, and the second domain of each of the second nucleic acid molecules hybridizes to about five third nucleic acid molecules, each of which comprises at least one detectable label. The first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule comprise L-DNA. A schematic of this exemplary reporter probe is shown in Figure 2. In some embodiments, the first domain of the first nucleic acid molecule is approximately 14 nucleotides long, the second domain of the first nucleic acid molecule is approximately 84 nucleotides long, the first domain of the second nucleic acid molecule is approximately 14 nucleotides long, the second domain of the second nucleic acid molecule is approximately 75 nucleotides long, and each of the third nucleic acid molecules is approximately 15 nucleotides long.

[0159] This disclosure provides a reporter probe comprising a first nucleic acid molecule comprising a first domain, a second domain, and a photocleavable linker placed between the first and second domains, wherein the second domain of the first nucleic acid molecule hybridizes to about six second nucleic acid molecules, each of the second nucleic acid molecules comprising a first domain, a second domain, and a photocleavable linker placed between the first and second domains, the first domain of each of the second nucleic acid molecules hybridizes to the second domain of the first nucleic acid molecule, the second domain of each of the second nucleic acid molecules hybridizes to about five third nucleic acid molecules, each of the third nucleic acid molecules comprising at least one detectable label, and the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule are composed of L-DNA. In some embodiments, the first domain of the first nucleic acid molecule is approximately 14 nucleotides long, the second domain of the first nucleic acid molecule is approximately 84 nucleotides long, the first domain of the second nucleic acid molecule is approximately 14 nucleotides long, the second domain of the aforementioned second nucleic acid molecule is approximately 75 nucleotides long, and each of the third nucleic acid molecules is approximately 15 nucleotides long.

[0160] In some embodiments, the photocleavable portion can be cleaved when exposed to UV light. The light can be provided by a light source selected from the group consisting of arc lamps, lasers, focused UV light sources, and light-emitting diodes.

[0161] The crackable linker portion is as follows: [ka] It can be a stereoisomer or salt thereof.

[0162] The crackable linker portion is [ka] It can be a stereoisomer or salt thereof.

[0163] The crackable linker portion is [ka] It can be.

[0164] The crackable linker portion is [ka] It can be.

[0165] The crackable linker portion is [ka] It can be.

[0166] In a preferred embodiment, the detectable label may be a fluorescent component or a fluorescent label. Those skilled in the art can refer to references directed toward labeling nucleic acids. Examples of fluorescent components include, but are not limited to, yellow fluorescent protein (YFP), green fluorescent protein (GFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, cyanines, dancylkloride, phycocyanin, and phycoerythrin.

[0167] Fluorescent labeling and their attachment to nucleotides and / or oligonucleotides are described in numerous reviews, including Haugland, Handbook of Fluorescent Probes and Research Chemicals, 9th edition (Molecular Probes, Inc., Eugene, 2002); Keller and Manak, DNA Probes, 2nd edition (Stockton Press, New York, 1993); Eckstein (ed.), Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991); and Wetmur, Critical Reviews in Biochemistry and Molecular Biology, 26:227-259 (1991). Specific methodologies applicable to this disclosure are disclosed in the following references: U.S. Patents No. 4,757,141, 5,151,507, and 5,091,519. For example, U.S. Patent No. 5,188,934 (4,7-dichlorofluorescein dye), No. 5,366,860 (spectrally resolvable rhodamine dye), No. 5,847,162 (4,7-dichlororhodamine dye), No. 4,318,846 (ether-substituted fluorescein dye), No. 5,800,996 (energy transfer dye), Lee et al., No. 5,066,580 (xanthine dye), No. 5,688,648 (energy transfer dye), etc. Labeling can also be carried out using quantum dots, as disclosed in the following patents and patent publications: U.S. Patents No. 6,322,901, 6,576,291, 6,423,551, 6,251,303, 6,319,426, 6,426,513, 6,444,143, 5,990,479, 6,207,392, 2002 / 0045045, and 2003 / 0017264. As used herein, the term “fluorescent labeling” encompasses a signaling portion that transmits information through the fluorescent absorption and / or luminescence properties of one or more molecules.These fluorescence properties include fluorescence intensity, fluorescence lifetime, emission spectral characteristics, and energy transition.

[0168] Commercially available fluorescent nucleotide analogs that readily integrate into nucleotide and / or oligonucleotide sequences include Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dUTP (Amersham Biosciences, Piscataway, New Jersey, USA), fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, Texas Red (TEXAS RED)-5-dUTP, Cascade Blue (CASCADE BLUE)-7-dUTP, BODIPY TMFL-14-dUTP, BODIPY TMR-14-dUTP, BODIPY TMTR-14-dUTP, Rhodamine Green (RHODAMINE GREEN)-5-dUTP, Oregon Green (OREGON GREEN)-488-5-dUTP, and Texas Red (TEXAS RED (trademark)-12-dUTP, BODIPY™ 630 / 650-14-dUTP, BODIPY™ 650 / 665-14-dUTP, ALEXA FLUOR (trademark) 488-5-dUTP, ALEXA FLUOR (trademark) 532-5-dUTP, Fluorescein-12-UTP, Tetramethylrhodamine-6-UTP, mCherry, Cascade Blue TM -7-UTP, BODIPY™ FL-14-UTP, BODIPY™ MR-14-UTP, BODIPY™ TR-14-UTP, Rhodamine Green™ (trademark)-5-UTP, ALEXA FLUOR TM (Trademark)488-5-UTP, ALEXA FLUOR TM(Trademarks) 546-14-UTP, ALEXA FLUOR (trademark) 594-5-UTP, ALEXA FLUOR (trademark) 546-14-dUTP (Molecular Probes, Inc., Eugene, Oregon, USA), etc. can be mentioned. Alternatively, the above fluorescent dyes (fluorophores) and those described in this specification can be added, for example, during oligonucleotide synthesis using phosphoramidite or NHS chemistry. Protocols for custom synthesis of nucleotides having other fluorescent dyes (fluorophores) are known in the art (see Henegariu et al. (2000) Nature Biotechnol. 18:345). 2-Aminopurine is a fluorescent base that can be directly incorporated into an oligonucleotide sequence during synthesis. Nucleic acids can also be stained a priori using intercalating dyes such as DAPI, YOYO-1, ethidium bromide, cyanine dyes (e.g., SYBR Green).

[0169] Other fluorescent dyes (fluorophores) available for attachment after synthesis are ALEXA FLUOR (trademark) 350, ALEXA FLUOR TM 405, ALEXA FLUOR TM 430, ALEXA FLUOR TM 532, ALEXA FLUOR TM 546, ALEXA FLUOR TM 568, ALEXA FLUOR TM 594, ALEXA FLUOR TM647, BODIPY 493 / 503, BODIPY FL, BODIPY R6G, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY TR, BODIPY 630 / 650, BODIPY 650 / 665, Cascade Blue, Cascade Yellow, Dansil, Lisamin Rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, Pacific Orange, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Tetramethylrhodamine, Texas Red (Molecular Probes, Inc., Eugene, This includes, but is not limited to, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, and Cy7 (Amersham Biosciences, Piscataway, New Jersey), which are available from Oregon. FRET tandem fluorescent dyes, including, but not limited to, PerCP-Cy5.5, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, APC-Cy7, PE-Alexa dyes (610, 647, 680), and APC-Alexa dyes, may also be used.

[0170] Metallic silver or gold particles can be used to enhance signals from fluorescently labeled nucleotide and / or oligonucleotide sequences (Lakowicz et al. (2003) BioTechniques 34:62).

[0171] Other suitable labels for oligonucleotide sequences may include fluorescein (FAM, FITC), digoxigenin, dinitrophenol (DNP), dansyl, biotin, bromodeoxyuridine (BrdU), hexahistidine (6xHis), and forformamino acids (e.g., P-tyr, P-ser, P-thr). The following hapten / antibody pairs can be used for detection, with each antibody being derivatized with a detectable label: biotin / α-biotin, digoxigenin / α-digoxigenin, dinitrophenol (DNP) / α-DNP, 5-carboxyfluorescein (FAM) / α-FAM.

[0172] The detectable labels described herein are spectrally resolvable. For multiple fluorescent labels, “spectrally resolvable” means that the fluorescence emission bands of the labels are sufficiently different, i.e., they do not overlap sufficiently, so that the molecular tags to which each label is attached can be identified by a standard photodetector, for example, using a bandpass filter and an optical intensifier tube, based on the fluorescence signal produced by each label, as exemplified by the system described in, for example, U.S. Patent No. 4,230,558, U.S. Patent No. 4,811,218, or Wheeles et al., pp. 21-76, Flow Cytometry: Instrumentation and Data Analysis (Academic Press, New York, 1985). Spectrally resolvable organic dyes, such as fluorescein and rhodamine, have maximum emission wavelengths located at least 20 nm apart, and in other embodiments, at least 40 nm apart. For chelated lanthanide compounds, quantum dots, etc., spectrally resolvable means that the maximum emission wavelengths are located at least 10 nm apart or at least 15 nm apart.

[0173] Imaging methods

[0174] This disclosure provides a method for detecting the abundance and spatial arrangement of multiple (two or more) target analytes in a biological sample using the ISH probe and reporter probe of this disclosure.

[0175] In some embodiments, the target analyte may be a nucleic acid (i.e., a target nucleic acid or target nucleic acid molecule). Accordingly, the Disclosure provides a method for detecting the abundance and spatial arrangement of multiple (two or more) target nucleic acids in a biological sample using the ISH probe and reporter probe of the Disclosure.

[0176] In some embodiments, the target analyte may be a protein (i.e., a target protein or target protein molecule). Accordingly, this disclosure provides a method for detecting the presence and spatial arrangement of multiple (two or more) target proteins in a biological sample using the ISH probe and reporter probe of this disclosure.

[0177] In some embodiments, the target analyte may be a carbohydrate molecule (e.g., a sugar moiety, a specific glycosylation motif, etc.). Accordingly, this disclosure provides a method for detecting the presence and spatial arrangement of multiple (two or more) target carbohydrate molecules.

[0178] The target nucleic acid can be any nucleic acid that the ISH probe of this disclosure can hybridize. The target nucleic acid may be DNA or RNA. In a preferred embodiment, the target nucleic acid is mRNA.

[0179] In short, each type of target nucleic acid to be detected in a biological sample is assigned a predefined unique ISH probe comprising: a) a target-binding domain complementary to that particular species of target nucleic acid (i.e., a target-binding domain designed to hybridize only to that particular species of target nucleic acid); and b) a unique barcode domain containing a unique nucleic acid sequence specific to the target nucleic acid of the said species. The unique nucleic acid sequence of the barcode domain is designed so that the specific sequence of the reporter probe of this disclosure binds sequentially to different attachment regions within the barcode domain, thereby constructing a “linear sequence of detectable labels” specific to the target nucleic acid of the said species.

[0180] The target protein can be any protein to which the ISH probe of this disclosure can bind.

[0181] In short, various target proteins to be detected in a biological sample are assigned a predefined unique ISH probe comprising: a) a target-binding domain that binds to the target protein of that particular species (i.e., is designed to bind only to the protein of that particular species); and b) a unique barcode domain containing a unique nucleic acid sequence specific to the protein of said species. The unique nucleic acid sequence of the barcode domain is designed so that the specific sequence of the reporter probe of this disclosure binds sequentially to different attachment regions within the barcode domain, thereby constructing a “linear sequence of detectable labels” specific to the target protein of said species.

[0182] The target carbohydrate molecule (sugar molecule) can be any carbohydrate molecule to which the ISH probe of this disclosure can bind. In some embodiments, the target carbohydrate molecule may be part of a specific glycosylated motif. In some embodiments, the target carbohydrate may be part of a specific lipid to be detected.

[0183] In short, each type of target carbohydrate to be detected in a biological sample is assigned a predefined unique ISH probe comprising: a) a target-binding domain that binds to a specific species of the target carbohydrate (i.e., designed to bind only to that specific species of carbohydrate); and b) a unique barcode domain containing a unique nucleic acid sequence specific to that species of carbohydrate. The unique nucleic acid sequence of the barcode domain is designed so that the specific sequence of the reporter probe of this disclosure binds sequentially to different attachment regions within the barcode domain, thereby constructing a "linear sequence of detectable labels" specific to that species of target carbohydrate.

[0184] An outline of non-limiting examples of these methods is shown in Figures 3A–3H, which illustrate the detection of two different target nucleic acids in a biological sample using the ISH probes and reporter probes of the Disclosure. The method begins with a biological sample containing two copies of target nucleic acid #1 (one in the upper left portion of the sample and the other in the lower right portion of the sample) and one copy of target nucleic acid #2 (in the upper right portion of the biological sample) in Figure 3A. In the first step of the method, the biological sample is brought into contact with several ISH probes of the Disclosure. An ISH probe having a target-binding domain complementary to target nucleic acid #1 (ISH probe type #1) hybridizes to target nucleic acid #1, and an ISH probe having a target-binding domain complementary to target nucleic acid #2 (ISH probe type #2) hybridizes to target nucleic acid #2. A third type probe (ISH probe type #3) having a target-binding domain complementary to a third type of target nucleic acid does not hybridize in the biological sample because the biological sample does not contain that third type of target nucleic acid.

[0185] In the second step, ISH probes that did not hybridize are washed out of the biological sample.

[0186] In the third step shown in Figure 3B, the biological sample is brought into contact with multiple reporter probes containing detectable labels. In this non-limiting example, the detectable labels are fluorescent labels. The barcode domain of ISH probe type #1 is designed so that the first attachment region hybridizes to a reporter probe having a red fluorescent label, and the barcode domain of ISH probe type #2 is designed so that the first attachment region hybridizes to a reporter probe having a green fluorescent label.

[0187] In the fourth step shown in Figure 3C, the attributes and spatial arrangement of the detectable labels of the hybridized reporter probe are recorded. Thus, during the first imaging, the red label was detected at "position 1", the green label at "position 2", and the red label at "position 3".

[0188] In the fifth step shown in Figure 3D, the detectable label is removed from the hybridized reporter probe. In this non-limiting example, the reporter probe includes a photocleavable portion that can be cleaved by UV light, which releases the detectable label, which is then washed away.

[0189] In step 6, shown in Figure 3E, the biological sample is brought into contact with a second group of reporter probes containing detectable labels. The barcode domain of ISH probe type #1 is designed so that the second attachment region hybridizes with a reporter probe having a yellow fluorescent label, and ISH probe type #2 is designed so that the second attachment region hybridizes with a reporter probe having a red fluorescent label.

[0190] In step 7, shown in Figure 3F, the attributes and spatial arrangement of the detectable labels of the hybridized reporter probe are recorded. Thus, during the second imaging, the yellow label was detected at position 1, the red label at position 2, and the yellow label at position 3.

[0191] In step 8, shown in Figure 3G, the detectable label is removed from the hybridized reporter probe by UV-induced cleavage of the photocleavable portion within the reporter probe.

[0192] These steps are repeated until all attachment regions within each ISH probe are bound by the reporter probe, and the detectable label identities of the reporter probes are sequentially recorded. Therefore, at the end of this method, the "linear order of detectable labels" will be recorded at each position of interest. As shown in Figure 3H, in this non-limiting example, the linear order of detectable labels at positions 1 and 3 is red-yellow-green-red, and the linear order of detectable labels at position 2 is green-red-yellow-yellow. Therefore, since red-yellow-green-red is specific to target nucleic acid #1 and green-red-yellow-yellow is specific to target nucleic acid #2, this method enables the identification of two copies of target nucleic acid #1 in a biological sample, with one copy present at position 1 and the other at position 3, and the identification of one copy of target nucleic acid #2 at position 2.

[0193] The above method can be multiplexed to detect any number of target nucleic acids and / or target proteins at any number of positions using a biological sample. In some embodiments, the method of the present disclosure can detect at least about 10, or at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, at least about 80, or at least about 90, or at least about 100, or at least about 110, or at least about 120, or at least about 130, or at least about 140, or at least about 150, or at least about 160, or at least about 170, or at least about 180, or at least about 19 in the biological sample. It can be used to determine the spatial abundance of 0, or at least about 200, or at least about 210, or at least about 220, or at least about 240, or at least about 250, or at least about 260, or at least about 270, at least about 280, at least about 290, or at least about 300, or at least about 500, or at least about 1,000, or at least about 10,000, or at least about 100,000, or at least about 1,000,000 different target nucleic acids and / or target proteins.

[0194] Accordingly, the present disclosure provides a method for determining the abundance and spatial arrangement of at least one target analyte in a biological sample, comprising: a) contacting a biological sample prepared according to the sample preparation method described herein with a plurality of reporter probes of the present disclosure, each reporter probe comprising at least one detectable label, thereby hybridizing the reporter probe to the attachment region of the barcode domain of at least one ISH probe bound to a target analyte in the biological sample; b) removing reporter probes that did not hybridize from the biological sample; c) recording the attributes and spatial arrangement of the detectable labels of the hybridized reporter probes; d) removing the detectable labels of the hybridized reporter probes; and e) repeating steps (a) to (d) until each attachment region within the barcode domain of the ISH probe bound to a target analyte in the biological sample is hybridized to a reporter probe comprising at least one detectable label, thereby determining the abundance and spatial arrangement of at least one target analyte in the biological sample based on the recorded sequence (order) of the detectable labels.

[0195] Accordingly, the present disclosure provides a method for determining the abundance and spatial arrangement of at least one target protein molecule in a biological sample, comprising: a) contacting a biological sample prepared according to the sample preparation method described herein with a plurality of reporter probes of the present disclosure, each reporter probe comprising at least one detectable label, thereby hybridizing the reporter probe to the attachment region of the barcode domain of at least one ISH probe bound to a target protein molecule in the biological sample; b) removing reporter probes that did not hybridize from the biological sample; c) recording the attributes and spatial arrangement of the detectable labels of the hybridized reporter probes; d) removing the detectable labels of the hybridized reporter probes; and e) repeating steps (a) to (d) until each attachment region within the barcode domain of an ISH probe bound to a target protein molecule in the biological sample is hybridized to a reporter probe comprising at least one detectable label, thereby determining the abundance and spatial arrangement of at least one target protein molecule in the biological sample based on the recorded sequence (order) of the detectable labels.

[0196] Accordingly, the present disclosure provides a method for determining the abundance and spatial arrangement of at least one target nucleic acid molecule in a biological sample, comprising: a) contacting a biological sample prepared according to the sample preparation method described herein with a plurality of reporter probes of the present disclosure, each reporter probe comprising at least one detectable label, thereby hybridizing the reporter probe to the attachment region of the barcode domain of at least one ISH probe hybridized to the target nucleic acid molecule in the biological sample; b) removing reporter probes that did not hybridize from the biological sample; and c) hybridizing The present invention provides a method for determining the abundance and spatial arrangement of at least one target nucleic acid molecule in a biological sample based on a sequence on which the detectable label is recorded, by repeating steps (a) to (d) until each attachment region within the barcode domain of the ISH probe hybridized to the target nucleic acid in the biological sample is hybridized to a reporter probe containing at least one detectable label.

[0197] Accordingly, the present disclosure provides a method for determining the abundance and spatial arrangement of at least one target analyte in a biological sample, comprising: a) contacting a biological sample prepared according to the sample preparation method described herein with a plurality of reporter probes of the present disclosure, each reporter probe comprising at least one detectable label, thereby hybridizing the reporter probe to the attachment region of the barcode domain of at least one ISH probe bound to the target analyte in the biological sample; b) removing reporter probes that did not hybridize from the biological sample; and c) recording the attributes and spatial arrangement of the detectable labels of the hybridized reporter probes. The present invention provides a method comprising: d) removing a detectable label from a hybridized reporter probe; and e) determining the abundance and spatial location of at least one target analyte in a biological sample based on the sequence (order) in which the detectable labels are recorded, by hybridizing each of at least one attachment region within the barcode domain of the ISH probe bound to the target analyte in the biological sample to a reporter probe containing at least one detectable label.

[0198] Accordingly, the present disclosure provides a method for determining the abundance and spatial arrangement of at least one target protein molecule in a biological sample, comprising: a) contacting a biological sample prepared according to the sample preparation method described herein with a plurality of reporter probes of the present disclosure, each reporter probe comprising at least one detectable label, thereby hybridizing the reporter probes to the attachment region of the barcode domain of at least one ISH probe bound to the target protein molecule in the biological sample; b) removing reporter probes that did not hybridize from the biological sample; c) recording the attributes and spatial arrangement of the detectable labels of the hybridized reporter probes; and d) recording the hybridized reporter probes. The present invention provides a method comprising the steps of: (a) removing a detectable label from a lobe; and (e) repeating steps (a) to (d) until each of the at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten attachment regions within the barcode domain of an ISH probe bound to a target protein molecule in the biological sample is hybridized to a reporter probe containing at least one detectable label, thereby determining the abundance and spatial arrangement of at least one target protein molecule in the biological sample based on the sequence (order) in which the detectable labels are recorded.

[0199] Accordingly, the present disclosure provides a method for determining the abundance and spatial arrangement of at least one target nucleic acid molecule in a biological sample, comprising: a) contacting a biological sample prepared according to the sample preparation method described herein with a plurality of reporter probes of the present disclosure, each reporter probe comprising at least one detectable label; b) removing reporter probes that did not hybridize from the biological sample; c) recording the attributes and spatial arrangement of the detectable labels of the hybridized reporter probes; d) removing the detectable labels of the hybridized reporter probes; and e) hybridizing to the target nucleic acid in the biological sample. The present invention provides a method comprising: repeating steps (a) to (d) until each of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten attachment regions within the barcode domain of an ISH probe is hybridized to a reporter probe containing at least one detectable label; and determining the abundance and spatial arrangement of at least one target nucleic acid molecule in the biological sample based on the sequence on which the detectable label is recorded.

[0200] Accordingly, the present disclosure provides a method for determining the abundance and spatial arrangement of at least two target analytes in a biological sample, comprising: a) contacting a biological sample prepared according to the sample preparation method described herein with a plurality of reporter probes of the present disclosure, each reporter probe comprising at least one detectable label; b) hybridizing the reporter probes to attachment regions within the barcode domain of at least one ISH probe bound to a target analyte in the biological sample; b) removing reporter probes that did not hybridize from the biological sample; c) recording the attributes and spatial arrangement of the detectable labels of the hybridized reporter probes; d) removing the detectable labels of the hybridized reporter probes; and e) repeating steps (a) to (d) until each attachment region within the barcode domain of an ISH probe bound to a target analyte in the biological sample is hybridized to a reporter probe comprising at least one detectable label, thereby determining the abundance and spatial arrangement of at least two target analytes in the biological sample based on the recorded sequence (order) of the detectable labels.

[0201] Accordingly, the present disclosure provides a method for determining the abundance and spatial arrangement of at least two target protein molecules in a biological sample, comprising: a) contacting a biological sample prepared according to the sample preparation method described herein with a plurality of reporter probes of the present disclosure, each reporter probe comprising at least one detectable label, thereby hybridizing the reporter probe to the attachment region of the barcode domain of at least one ISH probe bound to a target protein molecule in the biological sample; b) removing reporter probes that did not hybridize from the biological sample; c) recording the attributes and spatial arrangement of the detectable labels of the hybridized reporter probes; d) removing the detectable labels of the hybridized reporter probes; and e) repeating steps (a) to (d) until each attachment region within the barcode domain of an ISH probe bound to a target protein molecule in the biological sample is hybridized to a reporter probe comprising at least one detectable label, thereby determining the abundance and spatial arrangement of at least two target protein molecules in the biological sample based on the sequence on which the detectable labels are recorded.

[0202] Accordingly, the present disclosure provides a method for determining the abundance and spatial arrangement of at least two target nucleic acid molecules in a biological sample, comprising: a) contacting a biological sample prepared according to the sample preparation method described herein with a plurality of reporter probes of the present disclosure, each reporter probe comprising at least one detectable label; b) hybridizing the reporter probes to the attachment region of the barcode domain of at least one ISH probe hybridized to the target nucleic acid molecules in the biological sample; b) removing reporter probes that did not hybridize from the biological sample; c) hybridizing The present invention provides a method comprising: d) recording the attributes and spatial arrangement of a detectable label on a soyed reporter probe; e) removing the detectable label from a hybridized reporter probe; and e) repeating steps (a) to (d) until each attachment region within the barcode domain of an ISH probe hybridized to a target nucleic acid in the biological sample is hybridized to a reporter probe containing at least one detectable label, thereby determining the abundance and spatial arrangement of at least two target nucleic acid molecules in the biological sample based on the sequence (order) on which the detectable labels are recorded.

[0203] Accordingly, the present disclosure provides a method for determining the abundance and spatial arrangement of at least two target analytes in a biological sample, comprising: a) contacting a biological sample prepared according to the sample preparation method described herein with a plurality of reporter probes of the present disclosure, each reporter probe comprising at least one detectable label; b) removing reporter probes that did not hybridize from the biological sample; c) recording the attributes and spatial arrangement of the detectable labels of the hybridized reporter probes; d) removing the detectable labels of the hybridized reporter probes; and e) determining the abundance and spatial arrangement of the target analytes in the biological sample. The present invention provides a method comprising the step of determining the abundance and spatial arrangement of at least two target analytes in a biological sample based on the sequence (order) on which the detectable labels are recorded, by repeating steps (a) to (b) until each of the at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten attachment regions within the barcode domain of the ISH probe bound to the ISH probe has hybridized to a reporter probe containing at least one detectable label.

[0204] Accordingly, the present disclosure provides a method for determining the abundance and spatial arrangement of at least two target protein molecules in a biological sample, comprising: a) contacting a biological sample prepared according to the sample preparation method described herein with a plurality of reporter probes of the present disclosure, each reporter probe comprising at least one detectable label; b) hybridizing the reporter probes to the attachment region of the barcode domain of at least one ISH probe bound to a target protein molecule in the biological sample; b) removing reporter probes that did not hybridize from the biological sample; c) recording the attributes and spatial arrangement of the detectable labels of the hybridized reporter probes; and d) recording the hybridized reporter probes. The present invention provides a method comprising: e) removing a detectable label from the probe; and repeating steps (a) to (b) until each of the at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten attachment regions within the barcode domain of the ISH probe bound to a target protein molecule in the biological sample is hybridized with a reporter probe containing at least one detectable label, thereby determining the abundance and spatial arrangement of at least two target protein molecules in the biological sample based on the sequence (order) on which the detectable labels are recorded.

[0205] Accordingly, the present disclosure provides a method for determining the abundance and spatial arrangement of at least two target nucleic acid molecules in a biological sample, comprising: a) contacting a biological sample prepared according to the sample preparation method described herein with a plurality of reporter probes of the present disclosure, each reporter probe comprising at least one detectable label, thereby hybridizing the reporter probes to the attachment region of the barcode domain of at least one ISH probe hybridized to the target nucleic acid in the biological sample; b) removing reporter probes that did not hybridize from the biological sample; c) recording the attributes and spatial arrangement of the detectable labels of the hybridized reporter probes; and d) recording the hybridized reporter probes. The present invention provides a method comprising: e) removing a detectable label from a lobe; repeating steps (a) to (d) until each of the at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten attachment regions in the barcode domain of the ISH probe hybridized to the target nucleic acid in the biological sample is hybridized to a reporter probe containing at least one detectable label, thereby determining the abundance and spatial arrangement of at least two target nucleic acid molecules in the biological sample based on the sequence (order) on which the detectable labels are recorded.

[0206] In some embodiments of the above-described method, the step of determining the abundance and spatial arrangement of (one or more) target analytes may include defining one or more regions of interest within a biological sample using the abundance and spatial arrangement of the target analytes. In some embodiments, after defining one or more regions of interest within a tissue sample, the nucleic acid probe (i.e., ISH probe) bound to the target analyte can be removed from the biological sample, the biological sample can be re-contacted with at least one nucleic acid probe (i.e., ISH probe), and the above-described imaging method can be repeated only in the defined one or more regions of interest within the biological sample. While we do not wish to be bound by theory, defining one or more regions of interest within a biological sample allows for faster subsequent imaging steps because only specific regions of the tissue sample need to be searched and extracted (interrogated), as opposed to the entire tissue sample. In non-limiting examples, defining one or more regions of interest by determining the abundance and spatial arrangement of a target analyte allows a person skilled in the art to define tumorous areas of a biological specimen by performing the method described above, and to further analyze those tumorous areas of the biological specimen in subsequent imaging cycles. This method of defining one or more regions of interest is referred to herein as morphological scanning.

[0207] In some embodiments of the above-described method, the biological sample (specimen) may be a mounted biological sample prepared according to the sample processing method described herein. In some embodiments, the mounted biological sample is located in a flow cell prepared using the sample processing method described herein.

[0208] Any step of the sample preparation method described herein can be combined with any step of the imaging method described herein.

[0209] In some embodiments of the above-described method, the method may further include pre-treating the biological sample prior to step (a). In some embodiments, pre-treating the biological sample may include incubating the biological sample in a sulfo-NHS-acetic acid blocking solution. In some embodiments, pre-treating the biological sample may include washing the biological sample with a reporter washing buffer. In some embodiments, pre-treating the biological sample may include incubating the biological sample in an autofluorescence suppression buffer. In some embodiments, pre-treating the biological sample may include irradiating the biological sample with blue light and / or UV light to quench the autofluorescence of the sample via photobleaching. In some embodiments, the autofluorescence of the sample can be quenched via photobleaching using any combination of UV irradiation and readout channel irradiation.

[0210] In some embodiments, pretreatment of a biological sample may include i) incubating the biological sample in a sulfo-NHS-acetic acid blocking solution for about 15 minutes, and ii) washing the biological sample with a reporter washing buffer. In some embodiments, pretreatment of a biological sample may include i) incubating the biological sample for about 15 minutes, and ii) washing the biological sample with a reporter washing buffer, iii) incubating the biological sample in an autofluorescence suppressor buffer, and iv) washing the biological sample with a reporter washing buffer. In some embodiments, pretreatment of a biological sample may include i) incubating the biological sample for about 15 minutes, ii) washing the biological sample with a reporter washing buffer, iii) incubating the biological sample in an autofluorescence suppressor buffer and / or irradiating the biological sample with blue light and / or UV light to quench the autofluorescence of the sample via photobleaching, and iv) washing the biological sample with a reporter washing buffer.

[0211] In some embodiments, the step of washing the biological sample may include washing the biological sample with at least about 1000 mL of reporter washing buffer.

[0212] In some embodiments, the reporter washing buffer may include a 0.5% Tween-20 solution in a 1×SSPE solution.

[0213] In some embodiments, the autofluorescence suppression buffer may include any buffer that attenuates the autofluorescence of a tissue sample, as will be understood by those skilled in the art. A non-limiting example of an autofluorescence suppression buffer is TrueBlack® background suppressant solution (available from Biotium, Inc. Fremont, California, USA).

[0214] As will be understood by those skilled in the art, 20×SSPE buffer contains 0.02 M EDTA and 2.98 M NaCl in 0.2 M phosphate buffer pH 7.4.

[0215] In some embodiments, the sulfo-NHS-acetic acid blocking solution may contain a solution of 100 mM sulfo-NHS-acetic acid and 100 mM sodium phosphate at pH 8.0.

[0216] In some embodiments of the previous method, the step of contacting a biological sample with a plurality of reporter probes of the present disclosure includes incubating the biological sample with a certain solution for at least about 15 minutes, wherein the solution comprises at least one reporter probe at a concentration of 5 nM in DEPC-treated water, an 8.75×SSPE solution, 0.5% Tween-20, and a 0.1% RNase inhibitor.

[0217] In some embodiments of the above-described method, the step of contacting a biological sample with a plurality of reporter probes of the present disclosure may include incubating the biological sample with a solution for at least about 15 minutes, wherein the solution comprises at least one reporter probe at a concentration of 5 nM in DEPC-treated water, an 8.75×SSPE solution, 0.5% Tween-20, and optionally, a 0.1% RNase inhibitor.

[0218] In some embodiments of the above-described method, the step of removing reporter probes that did not hybridize from the biological sample may include washing the biological sample with reporter washing buffer. In some embodiments, the step of removing reporter probes that did not hybridize from the biological sample may include washing the biological sample with at least about 2000 mL of reporter washing buffer.

[0219] In some aspects of the above-described method, the step of recording the attributes and spatial arrangement of a detectable label of a hybridized reporter probe within a biological sample may include i) immersing the biological sample in an imaging buffer, and ii) imaging the biological sample to record the attributes and spatial arrangement of the detectable label of the hybridized reporter probe.

[0220] In some embodiments, the imaging buffer can match the refractive index of water, enabling imaging of a reference probe and a reporter probe without fading or attenuation of the fluorescence signal. In non-limiting examples, the imaging buffer can enable up to 4000 imagings per tissue location without fading or attenuation of the fluorescence signal.

[0221] The imaging buffers of this disclosure may include one or more of the following: pyranose oxidase, catalase, glucose oxidase, tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), 2-mercaptoethanol (BME), p-phenylenediamine (PPD), n-propyl gallate (NPG), 1,4-diazobicyclo[2.2.2]-octane (DABCO), ascorbic acid, 3-carboxy-PROXYL, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), N-tert-butyl-α-phenylnitrone, N-tert-butyl-α-(2-sulfophenyl)nitrone, 5,5-dimethyl-1-pyrroline N-oxide 4,4,4-Ethyl trifluorobutyrate, 4-Hydrazonomethyl-1-hydroxy-2,2,5,5-tetramethyl-3-imidazoline-3-oxide, α-(4-pyridyl N-oxide)-N-tert-butylnitrone, silver diethyldithiocarbamate, sodium diethyldithiocarbamate trihydrate, 3,3,5,5-tetramethyl-1-pyrroline-N-oxide, 1,3,5-tri-tert-butyl-2-nitrosobenzene, 2-(5,5-dimethyl-2-oxo-2λ5-[1,3,2]dioxaphosfinan-2-yl)-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (CYPMPO), vitamin E, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox;Trolox), N-acetyl-L-cysteine, 4-aminobenzohydrazide (myeloperoxidase inhibitor-I), balsalazid disodium salt hydrate, bilirubin, N-tert-butyl-α-phenylnitrone, caffeic acid, β-carotene, gallic acid catechin, chlorogenic acid, chlorophyllin sodium, p-coumaric acid, delphidin chloride, 5-O-(trans-3,4-dihydroxycinnamoyl)-D-quinic acid, DL-α-lipoic acid, ellagic acid, 2,4-Dihydro-5-methyl-2-phenyl-3H-pyrazole-3-one (MCI-186), (-)-epicatechin, (-)-epicatechin gallate, EUK-8, trans-ferric acid, 4-(5-fluoro-1H-indole-3-yl)butanamide, MPO inhibitor II (myeloperoxidase inhibitor-II), Fe(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride porphyrin pentachloride, Fe(III) 5,10,15,20-Tetrakis(4-sulfonatephenyl) polyphyllinate chloride, fucoxanthine carotenoid antioxidant, gallic acid, (-)-gallocatechin, ginkgolide, glutathione monoethyl ester, glutathione free acid, hesperidin, 3-hydroxytyrosol, 7-hydroxy-3-(4-methoxyphenyl)chromen-4-one (formonetin), kaempferol, linolein Acids, (±)-α-lipoic acid, luteolin, lycopene, L-lysine, Mn(III) tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP), meso-tetra(N-methyl-4-pyridyl)porfin tetratosylate salt (TMPyP), oleic acid, resveratrol, rutin hydrate, seleno-L-methionine, Se-(methyl)selenocysteine, sodium selenite, taxifolin hydrate, (+)-α-tocopherol, and xanthophyll.

[0222] In some embodiments, the imaging buffer may include a solution of 98% low-salt imaging buffer, 1% protocatechuic acid (PCA), and 1% protocatechuic acid dioxygenase (PCD). In some embodiments, the low-salt imaging buffer may include a solution of 1 M Tris-HCl pH 7.5, 5 M sodium chloride, and 0.5% Tween-20 in DEPC-treated water.

[0223] In some embodiments of the above-described method, the step of removing detectable labels from a hybridized reporter probe may include i) irradiating the biological sample with sufficient UV light to cleave the photocleavable linker portion in the hybridized reporter probe, and ii) washing the biological sample with reporter wash buffer. In some embodiments of the above-described method, removing detectable labels in a hybridized reporter probe may include i) irradiating the biological sample with sufficient UV light to cleave the photocleavable linker portion in the hybridized reporter probe, ii) washing the biological sample with reporter wash buffer, iii) immersing the biological sample in imaging buffer, and iv) imaging the sample to ensure that no detectable labels remain. In some embodiments, washing the biological sample with reporter wash buffer may include washing the biological sample with at least about 2000 mL of reporter wash buffer.

[0224] In some embodiments of the above-described method, removing a detectable label from a hybridized reporter probe may include i) irradiating the biological sample with sufficient UV light to cleave the photocleavable linker portion within the hybridized reporter probe, and ii) washing the biological sample with strip washing buffer. In some embodiments of the above-described method, removing a detectable label from a hybridized reporter probe may include i) irradiating the biological sample with sufficient UV light to cleave the photocleavable linker portion within the hybridized reporter probe, ii) washing the biological sample with strip washing buffer, iii) immersing the biological sample in imaging buffer, and iv) imaging the sample to ensure that no detectable label remains. In some embodiments, washing the biological sample with strip washing buffer may include washing the biological sample with at least about 2000 mL of strip washing buffer.

[0225] While not bound by theory, the combination of irradiating a biological sample with sufficient UV light to cleave the photocleavable linker portion in the hybridized reporter probe, and washing the biological sample with strip washing buffer, unexpectedly resulted in a more efficient and complete removal of all fluorescent labels, thereby eliminating potential fluorescence contamination from subsequent imaging cycles.

[0226] In some embodiments, washing a biological sample with reporter wash buffer may include washing at a flow rate of approximately 0.20 ml / min to approximately 0.55 ml / min, or approximately 0.60 ml / min to approximately 0.90 ml / min, or approximately 0.65 ml / min to approximately 0.85 ml / min, or approximately 0.70 ml / min to approximately 0.85 ml / min, or approximately 0.75 ml / min. In some embodiments, washing a biological sample with reporter wash buffer may include washing at a flow rate of approximately 0.75 ml / min. In some embodiments, washing a biological sample with reporter wash buffer may include washing at a flow rate of 0.20 ml / min to 0.55 ml / min, 0.60 ml / min to 0.90 ml / min, or 0.65 ml / min to 0.85 ml / min, or 0.70 ml / min to 0.85 ml / min, or 0.75 ml / min to 0.85 ml / min. In some embodiments, washing a biological sample with reporter washing buffer may include washing at a flow rate of 0.75 ml / min.

[0227] In some embodiments, washing a biological sample with a reporter washing buffer results in a flow rate of approximately 0.01 dyn / cm². 2 ~Approximately 20 dyn / cm 2 , or approximately 0.01 dyn / cm 2 ~Approximately 100 dyn / cm 2 , or approximately 8.89 dyn / cm 2 , or approximately 9 dyn / cm 2 This may include washing the sample so that there is shear stress on the sample plane. In some embodiments, washing the biological sample with a reporter washing buffer results in a wash of approximately 8.89 dyn / cm². 2 This may include washing the sample so that there is shear stress on the sample plane. In some embodiments, washing the biological sample with a reporter washing buffer may be 0.01 dyn / cm 2 ~20 dyn / cm 2 , or 0.01 dyn / cm 2 ~100 dyn / cm 2 , or 8.89 dyn / cm2 , or 9 dyn / cm 2 This may include washing the sample so that there is shear stress on the sample plane. In some embodiments, washing the biological sample with a reporter washing buffer may result in a pressure of 8.89 dyn / cm 2 This may include cleaning the sample so that there is shear stress on the surface of the sample.

[0228] In some embodiments, washing a biological sample with strip washing buffer may include washing at a flow rate of approximately 0.25 ml / min to approximately 0.55 ml / min, or approximately 0.3 ml / min to approximately 0.5 ml / min, or approximately 0.35 ml / min to approximately 0.45 ml / min, or approximately 0.4 ml / min. In some embodiments, washing a biological sample with strip washing buffer may include washing at a flow rate of approximately 0.4 ml / min. In some embodiments, washing a biological sample with strip washing buffer may include washing at a flow rate of 0.25 ml / min to 0.55 ml / min, or approximately 0.3 ml / min to 0.5 ml / min, or approximately 0.35 ml / min to 0.45 ml / min, or approximately 0.4 ml / min. In some embodiments, washing a biological sample with strip washing buffer may include washing at a flow rate of 0.4 ml / min.

[0229] In some embodiments, washing biological samples with strip washing buffer yields a rate of approximately 0.01 dyn / cm². 2 ~Approximately 20 dyn / cm 2 , or approximately 0.01 dyn / cm 2 ~Approximately 100 dyn / cm 2 , or approximately 8.89 dyn / cm 2 , or approximately 9 dyn / cm 2 This may include washing the sample so that there is shear stress on the sample plane. In some embodiments, washing the biological sample with strip washing buffer is approximately 8.89 dyn / cm 2This may include washing the sample so that there is shear stress on the sample plane. In some embodiments, washing the biological sample with strip washing buffer is performed at 0.01 dyn / cm 2 ~20 dyn / cm 2 , or 0.01 dyn / cm 2 ~100 dyn / cm 2 , or 8.89 dyn / cm 2 , or 9 dyn / cm 2 This may include washing the sample so that there is shear stress on the sample plane. In some embodiments, washing the biological sample with strip washing buffer is 8.89 dyn / cm 2 This may include cleaning the sample so that shear stress is present on the sample plane.

[0230] In some embodiments, any of the methods of the present disclosure may further include a morphological scan of a biological sample. In some embodiments, a morphological scan can be used to determine one or more regions of interest to be imaged. In some embodiments, a morphological scan can be used to determine distinctive properties of a biological sample (e.g., neoplastic cells, healthy cells, tumor excision margins, cell membranes, cell nuclei, one or more organelles, vascular structures, or any other properties known to those skilled in the art). In some embodiments, the distinctive properties of a biological sample may correlate with the abundance and spatial arrangement of a target analyte measured using the methods of the present disclosure. In some embodiments, a morphological scan can be used to define the contours of individual cells within a biological sample. The determination of the contours of individual cells is referred to herein as cell segmentation.

[0231] In some embodiments of the above method, the method may further include staining the biological sample with a membrane-specific fluorescent stain and imaging the biological sample to identify the spatial arrangement of cell membranes in the sample. This staining can be performed at any step of the protocol, for example, before contacting the attached biological sample with at least one nucleic acid probe, before contacting the biological sample prepared according to the sample preparation method described herein with the multiple reporter probes of this disclosure, after step (e), and so on.

[0232] In some embodiments of the methods described above, the method may further include staining a biological sample with a nuclear-specific fluorescent stain and imaging the biological sample to define the spatial arrangement of cell nuclei in the sample. This staining can be performed at any step of the protocol, for example, before contacting the mounted biological sample with at least one nucleic acid probe, before contacting the biological sample prepared according to the sample preparation method described herein with the multiple reporter probes of this disclosure, after step (e), and so on.

[0233] In some embodiments of the above-described method, the method may further include, after step (e), staining the biological sample with a membrane-specific fluorescent stain and imaging the biological sample to identify the spatial arrangement of cell membranes within the sample.

[0234] In some embodiments of the above-described method, the method may further include, after step (e), staining the biological sample with a nuclear-specific fluorescent stain and imaging the biological sample to identify the spatial arrangement of cell nuclei within the sample.

[0235] In some embodiments, membrane and / or nuclear staining is used to perform morphological scans on biological samples. Thus, in embodiments where membrane and / or nuclear staining is performed before contacting the biological sample with at least one nucleic acid probe or multiple reporter probes, membrane and / or nuclear staining can be used to define one or more regions of interest to be imaged during the determination of the abundance and spatial arrangement of target analytes (e.g., target nucleic acid molecules, target protein molecules, etc.). Without being bound by theory, since the total execution time of the experiment increases as the area of ​​the biological sample to be imaged increases, the total execution time of the imaging experiment can be reduced by imaging only specific regions of interest by identifying the area to be imaged using membrane and / or nuclear staining. Morphological scans using membrane and / or nuclear staining can be performed either before or after any step of any of the methods of the present disclosure, and can be repeated multiple times.

[0236] In some embodiments, the step of performing a morphological scan on a biological sample may include incubating the biological sample with an immunohistochemical blocking buffer. In some embodiments, the immunohistochemical blocking buffer may include buffer W. In some embodiments, the immunohistochemical blocking buffer may contain about 2% to about 5% BSA / BCS and about 0.5% Tween20 in about 8.75 × SSPE. In some embodiments, the immunohistochemical blocking buffer may contain 2% to 5% BSA / BCS and 0.5% Tween20 in 8.75 × SSPE.

[0237] In some embodiments, the step of performing a morphological scan on a biological sample may include contacting the biological sample with at least one probe. In some embodiments, the at least one probe may be an ISH probe of the present disclosure. In some embodiments, the at least one probe may be an antibody conjugated to a barcode domain as disclosed herein. In some embodiments, the at least one probe may be a lectin-targeted morphological protein conjugated to a barcode domain as disclosed herein.

[0238] In some embodiments, morphological scanning can be continued by contacting the biological sample with at least one probe, and then washing the biological sample with PBS to remove any unbound probes.

[0239] In some embodiments, after washing a biological sample with PBS to remove all unbound probes, morphological scanning can continue to visualize those probes (e.g., via binding of one or more reporter probes to the barcode domain). Visualizing the probes may comprise i) contacting a biological sample with a reporter probe described herein that hybridizes to the barcode domain of an ISH probe, ii) washing the biological sample with reporter washing buffer, iii) incubating the biological sample with imaging buffer, and iv) recording the attributes and spatial arrangement of the detectable label of the hybridized reporter probe. Visualizing the probes may further comprise repeating steps (i) to (iv) using similar steps described herein to remove the detectable label of the hybridized reporter probe and until each attachment site within the barcode domain is hybridized to the reporter probe.

[0240] In some embodiments of the above-described method, the method may further include, after step (e), f) washing the biological sample with strip washing buffer, g) immersing the biological sample in imaging buffer, h) imaging the biological sample to confirm that no detectable labels remain, j) incubating the biological sample with membrane staining blocking solution, k) washing the biological sample with reporter washing buffer, 1) immersing the biological sample in imaging buffer, m) imaging the biological sample to record the spatial arrangement of cell membranes in the biological solution, n) incubating the sample with nuclear staining solution, o) washing the biological sample with reporter washing buffer, p) immersing the sample in imaging buffer, and q) imaging the biological sample to record the spatial arrangement of cell nuclei in the sample.

[0241] In some embodiments of the above-described method, the method may further include, after step (e), f) washing the biological sample with strip washing buffer, g) immersing the biological sample in imaging buffer, h) imaging the biological sample to confirm that no detectable labels remain, i) incubating the sample with a nuclear staining solution, j) washing the sample with a reporter washing buffer, k) immersing the sample in imaging buffer, 1) imaging the biological sample to record the spatial arrangement of cell nuclei in the sample, m) incubating the biological sample with a membrane staining blocking solution, n) incubating the biological sample with a membrane staining solution, o) washing the biological sample with a reporter washing buffer, p) immersing the biological sample in imaging buffer, and q) imaging the biological sample to record the spatial arrangement of cell membranes in biological fluid.

[0242] In some embodiments of the above-described method, the method may further include, before or after any step: i) washing the biological sample with strip washing buffer; ii) immersing the biological sample in imaging buffer; iii) imaging the biological sample to confirm that no detectable labels remain; iv) incubating the biological sample with membrane staining blocking solution; v) incubating the biological sample with membrane staining solution; vi) washing the biological sample with reporter washing buffer; vii) immersing the biological sample in imaging buffer; viiii) imaging the biological sample to record the spatial arrangement of cell membranes in the biological fluid; ix) incubating the sample with nuclear staining solution; x) washing the biological sample with reporter washing buffer; xi) immersing the sample in imaging buffer; and xii) imaging the biological sample to record the spatial arrangement of cell nuclei in the sample.

[0243] In some embodiments of the above-described method, the method may further include, before or after any step: i) washing the biological sample with strip washing buffer; ii) immersing the biological sample in imaging buffer; iii) imaging the biological sample to confirm that no detectable labels remain; iv) incubating the sample with a nuclear staining solution; v) washing the sample with a reporter washing buffer; vi) immersing the sample in imaging buffer; vii) imaging the biological sample to record the spatial arrangement of cell nuclei in the sample; viiii) incubating the biological sample with a membrane staining blocking solution; ix) incubating the biological sample with a membrane staining solution; x) washing the biological sample with a reporter washing buffer; xi) immersing the biological sample in imaging buffer; and xii) imaging the biological sample to record the spatial arrangement of cell membranes in biological fluid.

[0244] In some embodiments, the biological sample can be incubated with the membrane staining blocking solution for at least about 30 minutes.

[0245] In some embodiments, the biological sample can be incubated with the membrane staining solution for at least about 60 minutes.

[0246] In some embodiments, the biological sample can be incubated with the nuclear stain for at least about 5 minutes. In some embodiments, the nuclear stain may contain 4',6-diamidino-2-phenylindole (DAPI) or DAPI dilactate. In some embodiments, the nuclear stain may contain about 100 nM to about 500 nM of DAPI diluted in PBS. In some embodiments, the nuclear stain may contain about 300 nM of DAPI diluted in PBS.

[0247] In some embodiments, the membrane staining solution may include a solution of 5% NaN3 and 1% DAPI in buffer W, and may further include at least one of the following: fluorescently labeled anti-CD298 antibody, fluorescently labeled anti-CD3 antibody, fluorescently labeled anti-CD20 antibody, and fluorescently labeled anti-PanCK antibody.

[0248] In some embodiments, the membrane staining solution may further include a solution in a membrane staining blocking solution comprising at least one of the following: fluorescently labeled anti-CD298 antibody, fluorescently labeled anti-B2M antibody, fluorescently labeled anti-CD3 antibody, fluorescently labeled anti-CD20 antibody, fluorescently labeled anti-PanCK antibody, fluorescently labeled anti-CD3 antibody, fluorescently labeled anti-histone H3 antibody, fluorescently labeled wheat germ agglutinin protein, and fluorescently labeled concanavalin A protein.

[0249] In some embodiments, the strip washing buffer may include a solution of 0.0033 × SSPE buffer and 0.5% Tween-20. In some embodiments, the strip washing buffer may include a solution of approximately 0.0033 × SSPE buffer and approximately 0.5% Tween-20.

[0250] In some embodiments, the strip washing buffer may include a solution of 0.0033 × SSPE buffer, 0.1% ProClin 950, and 0.5% Tween-20.

[0251] In some embodiments, the membrane staining blocking solution may include a solution of 0.5% NaN3 and 1% 4',6-diamidino-2-phenylindole (DAPI) in buffer W.

[0252] In some embodiments, the membrane staining blocking solution may include a solution of 0.5% NaN3 and 2 μg / mL of 4',6-diamidino-2-phenylindole (DAPI) in buffer W. In some embodiments, the membrane staining blocking solution may include a solution of approximately 0.5% NaN3 and approximately 2 μg / mL of 4',6-diamidino-2-phenylindole (DAPI) in buffer W.

[0253] In some embodiments, buffer W may contain at least one of fetal bovine serum (BCS), sodium azide (NaN3), dextran sulfate, and ssDNA. In some embodiments, buffer W may contain a combination of BCS, NaN3, dextran sulfate, and ssDNA. In some embodiments, the concentration of dextran sulfate in buffer W may be about 0.001% to about 0.1%, or about 0.005% to about 0.05%. In some embodiments, the concentration of dextran sulfate in buffer W may be about 0.01%. In some embodiments, the concentration of ssDNA in buffer W may be about 0.01 mg / ml to about 1 mg / ml, or about 0.05 mg / ml to about 0.5 mg / ml. In some embodiments, the concentration of ssDNA in buffer W may be about 0.1 mg / ml.

[0254] In some embodiments, the step of performing a morphological scan on a biological sample involves i) contacting the biological sample with at least one ISH probe, wherein the at least one ISH probe comprises a unique target-binding domain that binds to a target analyte in the biological sample and a unique barcode domain specific to the target analyte, the barcode domain comprising at least one attachment site; ii) contacting the prepared biological sample with a plurality of reporter probes, wherein each reporter probe comprises at least one detectable label, thereby hybridizing the reporter probe to the attachment site of the barcode domain of at least one ISH probe hybridized to the target analyte in the biological sample; and iii) The process may include: removing reporter probes that did not hybridize from the biological sample; iv) recording the attributes and spatial arrangement of the detectable labels of the hybridized reporter probes; v) removing the detectable labels of the hybridized reporter probes; and optionally, vi) repeating steps (ii) to (v) until each attachment site in the barcode domain of the ISH probe hybridized to the target analytes in the biological sample has hybridized to a reporter probe containing at least one detectable label, thereby determining the abundance and / or spatial arrangement of at least two target analytes in the biological sample based on the sequences on which the detectable labels are recorded, and thereby determining one or more regions of interest. In some embodiments of the above method, the target-binding domain may include an antibody. In some embodiments of the above method, the target-binding domain may include a lectin protein. In some embodiments of the above method, the barcode domain may include one attachment site.

[0255] In some aspects of the methods described above, as will be understood by those skilled in the art, a reference marker attached to the biological sample can be used to focus the biological sample using methods standard in the art. Specifically, the reference marker can be used to determine the best z-position for imaging a particular location within the biological sample.

[0256] kit

[0257] The present disclosure provides a kit for use in the methods of the present disclosure.

[0258] In some embodiments, the kits of the present disclosure can include any of the buffers and / or solutions described herein.

[0259] In some embodiments, the kits of the present disclosure can include multiple ISH probes of the present disclosure.

[0260] In some embodiments, the kits of the present disclosure can include multiple reporter probes of the present disclosure.

[0261] In some embodiments, the kits of the present disclosure can include an apparatus suitable for use in the methods of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0262] [Embodiment 1] A method for preparing a biological sample for fluorescence imaging, comprising: a) mounting a biological sample on a functionalized microscope slide to prepare a mounted biological sample, wherein the biological sample is a formalin-fixed paraffin-embedded (FFPE) microtome section; b) baking the mounted biological sample; c) dewaxing the mounted biological sample; d) performing a target activation reaction on the mounted biological sample; e) permeating the mounted biological sample; f) applying at least one reference marker to the mounted biological sample; g) fixing the mounted biological sample; h) contacting the mounted biological sample with at least one nucleic acid probe; and i) washing the mounted biological sample A method comprising the steps of.

[0263] [Embodiment 2] The method of Embodiment 1, further comprising assembling the attached biological sample into a flow cell after step (j).

[0264] [Embodiment 3] The method according to any of the above embodiments, wherein the functionalized microscope slide is a microscope slide functionalized with (3-aminopropyl)trimethoxysilane (APTMS).

[0265] [Embodiment 4] The method according to any of the above embodiments, wherein the biological sample is an FFPE microtome section of a human tissue sample.

[0266] [Embodiment 5] The method according to any of the above embodiments, wherein step (b) includes firing the attached biological sample at approximately 60°C for approximately 1 hour.

[0267] [Embodiment 6] The above step (c) is: i) Incubate the attached biological sample in a xylene solution for approximately 5 minutes; ii) Incubate the attached biological sample in a second xylene solution for approximately 5 minutes; iii) Incubate the attached biological sample in the first 100% ethanol solution for approximately 5 minutes; iv) Incubate the attached biological sample in the second 100% ethanol solution for approximately 2 minutes; and v) Dry the attached biological sample at approximately 60°C for approximately 5 minutes. A method according to any of the above embodiments, comprising:

[0268] [Embodiment 7] The above step (d) is: i) Incubate the attached biological sample in the target activation solution at approximately 100°C; ii) Incubate the attached biological sample in DEPC-treated water for approximately 15 seconds; iii) Incubate the attached biological sample in a 100% ethanol solution for approximately 3 minutes; and iv) Dry the attached biological sample. A method according to any of the above embodiments, comprising:

[0269] [Embodiment 8] The method of Embodiment 7, wherein the attached biological sample is incubated in the target activation solution for the time specified in Table 1.

[0270] [Embodiment 9] The method of Embodiment 7 or Embodiment 8, wherein the target activating solution comprises TRIS and EDTA solution and has a pH of about 9.

[0271] [Embodiment 10] The above step (e) is: i) Incubate the attached biological sample in a proteinase K solution at approximately 40°C, where the concentration of proteinase K in the proteinase K solution is approximately 1 μg / mL; ii) Wash the biological sample with the first aliquot of DEPC-treated water; and iii) Wash the biological sample with the DEPC-treated water from the second aliquot. A method according to any of the above embodiments, comprising:

[0272] [Embodiment 11] The method of Embodiment 11, comprising incubating the attached biological sample in a proteinase K solution for the time specified in Table 2.

[0273] [Embodiment 12] The above step (f) is: i) Incubating the attached biological sample for about 5 minutes in a solution containing at least one reference marker, wherein the solution containing at least one reference marker is a solution containing about 0.001% carboxylated microspheres and about 0.00045% uncarboxylated FND in a 2×SSC solution; and ii) Wash the mounted sample with 1×PBS. A method according to any of the above embodiments, comprising:

[0274] [Embodiment 13] The above step (g) is, (i) Incubating the mounted biological sample with 10% NBF for about 5 minutes; (ii) Incubating the mounted biological sample in a first Tris - glycine buffer solution for about 5 minutes; (iii) Incubating the mounted biological sample in a second Tris - glycine buffer solution for about 5 minutes; and (iv) Incubating the mounted biological sample in 1×PBS for about 5 minutes The method according to any of the above embodiments, comprising the above.

[0275] [Embodiment 14] The step (h) is: Incubating the mounted biological sample with a solution containing a plurality of ISH probes at about 37 °C for about 16 to about 18 hours, thereby hybridizing the at least one ISH probe to a target nucleic acid in the biological sample, where the solution contains at least two ISH probes, at least one of the ISH probes being present at a concentration of about 200 nM, The at least one ISH probe contains a unique target - binding domain that hybridizes to one of at least two target nucleic acids and a unique barcode domain specific to the target nucleic acid, the barcode domain containing at least four attachment sites. The method according to any of the above embodiments.

[0276] [Embodiment 15] The target - binding domain is a single - stranded polynucleotide containing a nucleic acid sequence complementary to the target nucleic acid, the target - binding domain is about 35 to about 40 nucleotides in length, the target - binding domain contains D - DNA, and the barcode domain is a single - stranded polynucleotide containing at least four attachment regions, where each of the attachment regions contains about one attachment sequence, each of the attachment sequences being about 14 nucleotides in length, and the sequences of each of the attachment sequences are different, and A method according to any of the above embodiments, wherein the barcode domain contains L-DNA.

[0277] [Embodiment 16] The above step (i) is: i) Incubate the attached biological sample with the first 2×SSC solution; ii) Incubate the attached biological sample in the first formamide solution; iii) Incubate the attached biological sample with the second formamide solution; iv) Incubate the attached biological sample with the second 2×SSC solution; and v) Incubate the attached biological sample with a third 2×SSC solution. A method according to any of the above embodiments, comprising:

[0278] [Embodiment 17] A method for determining the abundance and spatial arrangement of at least two target nucleic acid molecules in a biological sample, a) A step of contacting a biological sample prepared according to any one of the embodiments described above with a plurality of reporter probes of the present disclosure, each reporter probe comprising at least one detectable label, thereby hybridizing the reporter probe to the attachment region of the barcode domain of at least one ISH probe hybridized to a target nucleic acid in the biological sample; b) A step of removing the reporter probe that did not hybridize from the biological sample; c) A step of recording the attributes and spatial arrangement of the detectable labels of the hybridized reporter probe; d) Step of removing any detectable label from the hybridized reporter probe; e) Repeat steps (a) to (d) until each of the at least four attachment sites within the barcode domain of the ISH probe hybridized to the target nucleic acid in the biological sample has hybridized to a reporter probe containing at least one detectable label; This process involves determining the abundance and spatial arrangement of at least two target nucleic acid molecules in a biological sample based on a sequence on which detectable labels are recorded. A method according to any of the embodiments described above, including the method described above.

[0279] [Embodiment 18] The reporter probe is: A first nucleic acid molecule comprising a first domain, a second domain, and a photocleavable linker placed between the first and second domains, The second domain of the first nucleic acid molecule hybridizes to approximately six second nucleic acid molecules. Each of the second nucleic acid molecules comprises a first domain, a second domain, and a photocleavable linker placed between the first and second domains. Each of the first domains of the second nucleic acid molecule hybridizes to the second domain of the first nucleic acid molecule. Each of the second domains of the aforementioned second nucleic acid molecule hybridizes into approximately five third nucleic acid molecules. Each of the third nucleic acid molecules comprises at least one detectable label, and The first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule each contain L-DNA. The method of Embodiment 17.

[0280] [Embodiment 19] The method of Embodiment 17 or 18, wherein the at least one detectable label is a fluorescent portion.

[0281] [Embodiment 20] The above method, before step (a), i) Incubate the biological sample in a sulfo-NHS acetate blocking solution for approximately 15 minutes; ii) Wash the biological sample with reporter washing buffer; iii) Incubate the biological sample in autofluorescence suppression buffer; and iv) Wash the biological sample with reporter washing buffer. A method which further includes any one of embodiments 17, 18, or 19.

[0282] [Embodiment 21] A method according to any one of Embodiments 17, 18, 19, or 20, wherein step (a) comprises incubating a biological sample with a solution containing a reporter probe at a concentration of 5 nM in DEPC-treated water, an 8.75 × SSPE solution, 0.5% Tween-20, and a 0.1% RNase inhibitor for at least about 15 minutes.

[0283] [Embodiment 22] A method according to any one of Embodiments 17, 18, 19, 20, or 21, wherein step (b) comprises washing the biological sample with reporter washing buffer.

[0284] [Embodiment 23] A method of any one of Embodiments 17, 18, 19, 20, 21 or 22, wherein step (c) comprises i) immersing a biological sample in an imaging buffer; and ii) imaging the biological sample to record the attributes and spatial arrangement of a detectable label of a hybridized reporter probe.

[0285] [Embodiment 24] The above step (d) is: i) Irradiate the biological sample with sufficient UV light to cleave the photocleavable linker portion in the hybridized reporter probe; ii) Wash the biological sample with reporter washing buffer; iii) Immerse the biological sample in the imaging buffer; and iv) Image the sample to confirm that no detectable labels remain. One of the methods of Embodiments 17, 18, 19, 20, 21, 22, or 23, including the method described above.

[0286] [Embodiment 25] The method according to any one of Embodiments 17, 18, 19, 20, 21, 22, 23, or 24, further comprising, after step (e), staining the biological sample with a membrane-specific fluorescent stain and imaging the biological sample to identify the spatial arrangement of cell membranes within the sample.

[0287] [Embodiment 26] The method according to any one of Embodiments 17, 18, 19, 20, 21, 22, 23, 24, or 25, further comprising, after step (e), staining the biological sample with a nuclear heterofluorescence stain and imaging the biological sample to identify the spatial arrangement of cell nuclei in the sample. [Examples]

[0288] Example 1a

[0289] The following non-limiting examples describe sample preparation protocols for use in the methods of this disclosure.

[0290] First, 5 μm FFPE microtome sections of tissue were mounted onto a first microscope slide functionalized with 0.5% (3-aminopropyl)trimethoxysilane. As will be understood by those skilled in the art, the first microscope slide was functionalized using methods standard in the industry. As part of sample preparation, the first microscope slide will serve as one surface of a flow cell to be assembled later.

[0291] Next, the slides were baked in a 60°C oven for 1 hour. After baking, the paraffin on the slides was removed using xylene. Then, the pre-fixation with paraformaldehyde was removed by heating the slides at 100°C for 8 minutes in a target retrieval solution (e.g., 10 mM Tris, 1 mM EDTA at pH 9.0). The heating time can be adjusted to suit different sample types (e.g., tissue, cell pellets). In non-limiting cases, the slides can be heated for 8 minutes in the case of cell pellets.

[0292] After heating, a mixture of 0.001% red, blue, and green 200 nm carboxylated microspheres and 0.00045% non-carboxylated fluorescent nanodiamonds (FND) was applied to the tissue, incubated for 5 minutes, and then washed with 1× phosphate buffer solution (PBS).

[0293] Without being constrained by theory, the mixture of the reference sample and FND is used in subsequent imaging processes for autofocus and image registration.

[0294] Following the addition of the reference sample and FND, the tissue samples were post-fixed in 10% neutral buffered formalin (NBF) for 5 minutes. Without being bound by theory, this post-fixation step is used to preserve the morphology of the tissue samples. The NBF was then neutralized with Tris-glycine buffer for 10 minutes and subsequently washed with 1×PBS.

[0295] Next, the in-situ hybridization (ISH) probe of this disclosure was denatured at 95°C for 2 minutes, cooled on ice, and then applied to a tissue sample at a concentration of 0.5 nM per probe in a hybridization mixture containing Buffer R solution (stock solution: 3.125% dextran sulfate, 0.25% BSA, 0.125 mg / mL ssDNA, 2.5×SSC, 50% formamide; concentration used: 2.5% dextran sulfate, 0.2% BSA, 0.1 mg / mL ssDNA, 2×SSC, 40% formamide) and an RNase inhibitor. The tissue sample was incubated with the ISH probe at 37°C for 16-18 hours.

[0296] Following incubation with the ISH probe, the slides were briefly immersed in 2× saline-sodium citrate (SSC) containing 0.1% Tween-20 solution, then incubated for at least 50 minutes with two changes of 50% formamide and 2× SSC solution to wash away any excess unbound ISH probe. The slides were then optionally dehydrated by an ethanol gradient using the industry standard method, as will be understood by those skilled in the art. After dehydration, the tissue samples, if any, could be stored at 4°C for later use or immediately assembled into a flow cell for subsequent imaging steps.

[0297] Example 1b

[0298] In the following non-limiting examples, sample preparation protocols for use in the methods of this disclosure are described.

[0299] First, 5 μm FFPE microtome sections of tissue were mounted onto a first microscope slide functionalized with 0.5% (3-aminopropyl)trimethoxysilane. This first microscope slide was functionalized using a method standard in the art, as will be understood by those skilled in the art. As part of the sample preparation, the first microscope slide will serve as one surface of a flow cell to be assembled thereafter.

[0300] Next, the slides were baked in a 60°C oven for 1 hour. After baking, the paraffin on the slides was removed using xylene. Then, the paraformaldehyde pre-fixation was removed (undo) by heating in a target retrieval solution (e.g., 10 mM Tris, 1 mM EDTA at pH 9.0) at 100°C for 8 minutes. The heating time can be adjusted to suit different sample types (e.g., tissue, cell pellets, etc.). In non-limiting cases, for cell pellets, the slides can be heated for 8 minutes.

[0301] After heating, solutions of 0.0005%–0.003% 200 nm carboxylated microspheres stained red, yellow, blue, and green were applied to the tissue, incubated for 5 minutes, and then washed with 1× phosphate buffer (PBS).

[0302] Without being constrained by theory, a mixture of the reference sample and FND is used in subsequent imaging processes for autofocus and image registration.

[0303] Following the addition of the reference material, the tissue samples were post-fixed in 10% neutral buffered formalin (NBF) for 5 minutes. Without being bound by theory, this post-fixation step is used to preserve the morphology of the tissue samples. The NBF was then neutralized with Tris-glycine buffer and washed with 1×PBS.

[0304] Next, the in-situ hybridization (ISH) probe of this disclosure was denatured at 95°C for 2 minutes, cooled on ice, and then applied to a tissue sample at a concentration of 0.5 nM per probe in a hybridization mixture containing Buffer R solution (stock solution: 3.125% dextran sulfate, 0.25% BSA, 0.125 mg / mL ssDNA, 2.5×SSC, 50% formamide; concentration used: 2.5% dextran sulfate, 0.2% BSA, 0.1 mg / mL ssDNA, 2×SSC, 40% formamide) and an RNase inhibitor. The tissue sample was incubated with the ISH probe at 37°C for 16-18 hours.

[0305] Following incubation with the ISH probe, the slides were briefly immersed in 2× saline-sodium citrate (SSC) containing 0.1% Tween-20 solution, then incubated for at least 50 minutes with two changes of 50% formamide and 2× SSC solution to wash away any excess unbound ISH probe. The slides were then optionally dehydrated by an ethanol gradient using the industry standard method, as will be understood by those skilled in the art. After dehydration, the tissue samples, if any, could be stored at 4°C for later use or immediately assembled into a flow cell for subsequent imaging steps.

[0306] Example 2

[0307] In the following non-limiting examples, sample preparation protocols for use in the methods of this disclosure are described.

[0308] Day 0 – Before proceeding with the sample preparation protocol, the microscope slides could be functionalized using the following protocol. First, as will be understood by those skilled in the art, the microscope slides were cleaned using a plasma apparatus in a manner standard in the art. After cleaning, the slides were immersed in a 0.5% (3-aminopropyl)trimethoxysilane solution for 1 minute. After immersion, the slides were sonicated in a 0.5% (3-aminopropyl)trimethoxysilane solution for 10 seconds. The immersion and sonication were repeated twice, with the total time the slides spent in the 0.5% (3-aminopropyl)trimethoxysilane solution being approximately 3.5 minutes. The slides were then rinsed with water at least 3-4 times. Finally, the slides were dried under nitrogen.

[0309] Before sample preparation, biological samples (e.g., tissue samples) can be sectioned for use in the method of this disclosure. Biological samples were cut into 5 μm FFPE microtome sections and mounted on functionalized slides (see above). The slides with the mounted microtome sections were then dried overnight at room temperature. After drying, if the slides were not intended for immediate processing, they were stored at 4°C.

[0310] Day 1 – The slides with the microtome sections mounted (see above) were first fired at 60°C for 1 hour.

[0311] Paraffin-free

[0312] After firing, the slides were immediately transferred to a xylene solution and incubated for 5 minutes with stirring. Then, the slides were transferred to a fresh xylene solution and incubated for another 5 minutes with stirring. Next, the slides were transferred to a 100% ethanol solution and incubated for 2 minutes. Then, the slides were transferred to a fresh 100% ethanol solution and incubated for another 2 minutes. After this incubation, the slides were laid flat in a 60°C oven and dried for 2 minutes.

[0313] Targeted activation

[0314] Next, the 1× Target Activation Solution (prepared using diethyl pyrocarbonate (DEPC) treated water) was preheated to 100°C. For example, the solution can be preheated using a pressure cooker. The 1× Target Activation Solution should not be boiled for more than 15 minutes. Once the 1× Target Activation Solution reached 100°C, the slides were incubated in the solution for a period corresponding to the type of sample being treated. Incubation times for different sample types are shown in Table 1. After incubation at 100°C, the slides were immediately transferred to DEPC-treated water and incubated for 15 seconds with stirring. The slides were then transferred to a 100% ethanol solution and incubated for 3 minutes. The slides were then removed from the ethanol solution and allowed to dry for 5 minutes.

[0315] Tissue permeabilization (clearance treatment) and application of reference material / FND

[0316] After the targeted activation step, a hydrophobic barrier was drawn around the sample mounted on the slide, for example, using a PAP pen. Care was taken to ensure that the barrier was not too close to the tissue.

[0317] After marking the hydrophobic barrier, a 1 μg / mL proteinase K solution in PBS was prepared. The slides were then placed on a humidified tray lined with paper moistened with DEPC water and heated at 40°C for at least 30 minutes. Once the slides were in the humidified tray, the proteinase K solution was applied to the biological sample mounted on the slide. The slides were placed in a 40°C oven and incubated according to the type of biological sample. Incubation times for different sample types are shown in Table 2. After incubation, the proteinase K solution was removed from the biological sample. The slides were then washed 3–6 times with DEPC-treated water while agitating. Fresh DEPC-treated water was used for at least the final washing step.

[0318] After washing the slides, the reference / FND mixture, vortex-mixed for 30 seconds, was applied to the biological samples on the slides. An exemplary reference / FND mixture can be prepared by diluting 0.001% 200 nm carboxylated microsphere references, stained red, blue, and green, to 0.001% in a 2× saline-sodium citrate (SSC) solution, and then diluting non-carboxylated fluorescent nanodiamonds (FND) to 0.00045% in the same solution. This solution was then vortex-mixed for 1 minute, sonicated for 2 minutes, vortex-mixed again for 1 minute, and then sonicated again for 2 minutes. The reference / FND mixture was incubated with the biological samples at room temperature for 5 minutes. The slides were then washed with 1× PBS.

[0319] Post fixing

[0320] Following tissue permeabilization and application of reference material / FND, the slides were incubated in 10% neutral buffered formalin (NBF) for 5 minutes. Next, the slides were incubated in Tris-glycine buffer for 5 minutes. Then, the slides were incubated in a fresh batch of Tris-glycine buffer for 5 minutes. Finally, the slides were incubated in 1×PBS for 5 minutes.

[0321] Hybridization of the ISH probe of this disclosure

[0322] Next, the in-situ hybridization (ISH) probe of this disclosure was denatured at 95°C for 2 minutes and applied to the tissue sample for 1 minute in a hybridization mixture containing buffer R solution (stock solution), followed by cooling on ice for 1 minute; (3.125% dextran sulfate, 0.25% BSA, 0.125 mg / mL ssDNA, 2.5×SSC, 50% formamide; concentration used: 2.5% dextran sulfate, 0.2% BSA, 0.1 mg / mL ssDNA, 2×SSC, 40% formamide), and an RNase inhibitor. The tissue sample was incubated with the ISH probe for 16-18 hours. After pre-washing with the RNase inhibitor, the sample was incubated at 37°C in a container lined with paper moistened with 2×SSC solution.

[0323] Stringent cleaning

[0324] After hybridization of the ISH probe, the slides were removed from the oven and briefly immersed in 2×SSC solution. The slides were then incubated for 25 minutes in 2×SSC solution with 50% formamide (formamide preheated to 37°C), and after preheating the formamide to 37°C, the slides were incubated for 25 minutes in fresh aliquots of 50% formamide in 2×SSC solution. The slides were then incubated in 2×SSC solution for 2 minutes. Finally, the slides were incubated for another 2 minutes in fresh aliquots of 2×SSC solution.

[0325] Any dehydration

[0326] After stringent washing of the slides, they were optionally dehydrated with an ethanol gradient. The slides were first incubated in a 70% ethanol solution for 3 minutes, then in an 85% ethanol solution for 3 minutes, and finally in a 100% ethanol solution for 3 minutes.

[0327] After dehydration, the slides, if any, are immediately assembled into a flow cell used in the imaging method of this disclosure, or stored at 4°C for later use.

[0328] Example 3

[0329] The following non-limiting examples illustrate a flow cell assembly protocol using slides prepared in Example 1 or Example 2 for use in the methods of the present disclosure.

[0330] First, a 300 μm thick coverslip was washed with isopropanol to remove dust, debris, and / or water. Next, a 75 μm thick flow cell adhesive was applied to the coverslip. Then, the slide containing the biological sample (prepared as described in Example 1 or Example 2) was washed with isopropanol. Isopropanol is used to wipe around the mounted biological sample with a wipe to remove dust and / or water. If the sample is not dehydrated, a Kimwipe or suitable alternative is used to wipe around the mounted biological sample until the slide is liquid-free. If the biological sample appears completely dry, care was taken to ensure that the biological sample was moist, including by applying an appropriate buffer. Next, a flow cell was prepared by sliding the adhesive-coated coverslip with the mounted biological sample using a hydraulic press at a pressure of, for example, 250 psi for at least 30 seconds. Then, the coverslip was washed with isopropanol.

[0331] Example 4

[0332] The following non-limiting examples illustrate various solutions that can be used in the methods of this disclosure.

[0333] Sulfo-NHS acetate blocking solution: 100 mM sulfo-NHS acetate in 100 mM sodium phosphate, pH 8.0.

[0334] Reporter probe solution: 5 nM reporter probe of the present disclosure in DEPC-treated water, 8.75 × SSPE buffer, 0.5% Tween-20, 0.1% RNase inhibitor.

[0335] Low-salt imaging buffer: 1 M Tris-HCl pH 7.5, 5 M sodium chloride and 0.5% Tween-20 in DEPC-treated water.

[0336] Imaging buffer: 98% low-salt imaging buffer, 1% protocatechuic acid (PCA), and 1% protocatechuic acid dioxygenase (PCD).

[0337] Membrane staining blocking solution: 0.5% NaN3 and 1% 4',6-diamidino-2-phenylindole (DAPI) in buffer W.

[0338] Membrane staining solution: 5% NaN3 and 1% DAPI in buffer W, containing at least one of the following: fluorescently labeled anti-CD298 antibody, fluorescently labeled anti-CD3 antibody, fluorescently labeled anti-CD20 antibody, and fluorescently labeled anti-Panck antibody.

[0339] Reporter washing buffer: 0.5% Tween-20 in 1×SSPE solution.

[0340] Strip washing buffer: 0.0033 × SSPE buffer and 0.5% Tween-20.

[0341] Example 5

[0342] The following are non-limiting examples of the analysis of biological samples using the sample preparation and imaging methods of this disclosure.

[0343] Samples containing various cell lines such as CCRF-CEM cells, SUDHL4 cells, MDA-MB-468 cells, HS578T cells, EKVK cells, HCT116 cells, HOP92 cells, and COLO205 cells, as well as various FFPE samples and other biological samples, were prepared as described in the above examples. Subsequently, the target analytes in the biological samples were analyzed using the imaging method described herein by sequentially attaching reporter probes to ISH probes bound to the target analytes.

[0344] For control, abundance measurements obtained using the method of this disclosure were compared with known abundance data collected using standard RNA-Seq sequencing techniques. As shown in Figure 4, nucleic acid abundance data measured using the method of this disclosure showed high agreement with standard RNA-Seq data for genes above the detection limit (defined as >1 FPKM expression level in the Cancer Cell Encyclopedia Database), demonstrating sensitivity and specificity comparable to that of standard RNA-Seq techniques. While we do not wish to be bound by theory, these results demonstrate that the method of this disclosure accurately measures the abundance of a target analyte while adding the advantage, unlike standard RNA-Seq, that the spatial aspects of the target analyte are preserved and recorded.

[0345] Figure 5 shows images of individual target analytes detected in a biological sample containing MDA-MB-468 cells, including specific target analytes EEF1Al, MALAT1, and H4C3. Signals recorded from the negative probe (NegPrb 6) are also included. The graphs and tables in Figure 5 also show the number of cells in which a particular number of transcripts were detected using the method of this disclosure. As shown in Figure 5, at least 100 transcripts were detected in more than 97% of the cells, with a median transcript count of 1265 per cell. Furthermore, Figure 5 shows that the method of this disclosure was able to individually segment 3257 cells in the biological sample analyzed. Without being constrained by theory, the results shown in Figure 5 demonstrate that the method of this disclosure can determine the abundance and spatial arrangement of individual target analytes in biological samples at subcellular resolution, including very abundant target analytes (e.g., EEF1A1 in Figure 5), moderately abundant target analytes (MALAT1 in Figure 5), and rare transcripts (e.g., H4C2 in Figure 5).

[0346] Figure 6A shows an image of an FFPE melanoma tissue sample analyzed according to the method of this disclosure. In this experiment, 1,000 different target analytes were measured and spatially detected at subcellular resolution. Specifically, 22 negative probes and 997 ISH probes targeting specific target nucleic acids were used without being constrained by theory. The ability of the method of this disclosure to determine the spatial abundance of 1,000 target analytes in a target tissue sample enables comprehensive spatial single-cell analysis of the tissue sample, including cell typing and mapping, identification of cellular state, identification of cellular function, interaction analysis, differential expression analysis, and hormone activity analysis, as shown in the drawings. Figures 6B and 6C show the method of this disclosure performed on additional FFPE samples, including non-small cell lung cancer (NSCLC) FFPE samples, renal cell carcinoma FFPE samples, colorectal cancer (CRC) FFPE samples, and tonsil FFPE samples. Cell typing results mapped to the tissue pieces are shown in Figures 6D–6G.

[0347] The results described in this embodiment demonstrate that the method of this disclosure enables the simultaneous quantification of the spatial abundance of thousands of target analytes in biological samples such as tissue samples, thereby allowing those skilled in the art to perform a wide variety of biological analyses at the single-cell level.

[0348] The doctrine of equality

[0349] The above description is provided for illustrative purposes only and is not intended to limit the disclosure to the exact form disclosed. Details of one or more embodiments of the disclosure are described in the accompanying description above. Any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the disclosure, but preferred methods and materials are described. Other features, purposes and advantages of the disclosure will become apparent from the specification and claims. In this specification and the accompanying claims, singular forms also include plural forms unless the context explicitly indicates otherwise. All technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which this disclosure belongs, unless otherwise defined. All patents and publications cited herein are incorporated herein by reference.

Claims

1. A method for determining the abundance and spatial arrangement of at least two target analytes in a biological sample, The aforementioned biological sample i) Incubating the attached biological sample with a solution containing multiple in situ hybridization (ISH) probes, thereby bringing the biological sample into contact with at least one nucleic acid probe, The aforementioned solution contains at least two types of ISH probes, The at least one ISH probe comprises a unique target-binding domain that binds to one of the at least two target analytes, and a unique barcode domain specific to the one target analyte, wherein the barcode domain includes at least one attachment region, and the contact; and ii) Washing the attached biological sample. Prepared by, The method described above is a) A step of bringing a prepared biological sample into contact with a plurality of reporter probes, each of which includes at least one detectable label, thereby hybridizing the reporter probe to the attachment region of the barcode domain of at least one ISH probe hybridized to the target analyte in the prepared biological sample; b) A step of removing the reporter probe that did not hybridize from the prepared biological sample; c) A step of recording the attributes and spatial arrangement of at least one detectable label of the hybridized reporter probe; d) the step of removing at least one detectable label from the hybridized reporter probe; and e) Repeating steps (a) to (d) until each attachment region within the barcode domain of the ISH probe hybridized to the target analytes in the prepared biological sample hybridizes to a reporter probe containing at least one detectable label, thereby recording a specific sequence of hybridized reporter probes sequentially bound to different attachment regions within the barcode domain, identifying at least two target analytes based on the recorded specific sequence, and determining the abundance and spatial arrangement of at least two target analytes in the biological sample; The method, including the method described above.

2. The above-mentioned at least two target analytes are target nucleic acid molecules, The target-binding domain is a single-stranded polynucleotide containing a nucleic acid sequence complementary to the target nucleic acid. The target-binding domain is 35 to 40 nucleotides long, The aforementioned target-binding domain contains D-DNA, and, The barcode domain is a single-stranded polynucleotide comprising at least one attachment region, Here, each of the aforementioned attachment regions includes at least one attachment sequence, each of the aforementioned attachment sequences is 14 nucleotides long, and each of the aforementioned attachment sequences has a different sequence, and The method according to claim 1, wherein the barcode domain contains L-DNA.

3. The above at least two target analytes are target protein molecules, and The method according to claim 1, wherein the target-binding domain comprises an antibody or antigen-binding fragment that specifically binds to the target protein molecule.

4. The aforementioned barcode domain, i) at least two, ii) At least three, iii) at least four, or iv) at least 5 The method according to any one of claims 1 to 3, comprising an attachment region.

5. The method according to claim 1, wherein the solution comprises at least one negative ISH probe designed not to specifically bind to any target analyte in the biological sample.

6. The method according to claim 5, wherein the negative ISH probe comprises at least one External RNA Standard Consortium (ERCC) evaluation sequence or its complement.

7. The method according to any one of claims 1 to 6, wherein the reporter probe comprises L-DNA.

8. The reporter probe comprises a first nucleic acid molecule comprising a first domain, a second domain, and a photocleavable linker placed between the first and second domains. Here, the second domain of the first nucleic acid molecule hybridizes into six second nucleic acid molecules, Each of the second nucleic acid molecules comprises a first domain, a second domain, and a photocleavable linker placed between the first domain and the second domain. Here, each of the first domains of the second nucleic acid molecule hybridizes to the second domain of the first nucleic acid molecule, Each of the second domains of the aforementioned second nucleic acid molecule hybridizes into five third nucleic acid molecules. Each of the third nucleic acid molecules comprises at least one detectable label, and The first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule each contain L-DNA. The method according to any one of claims 1 to 7.

9. The method according to any one of claims 1 to 8, wherein the at least one detectable label is a fluorescent portion.

10. The above method, before step (a), i) Incubate the biological sample in N-hydroxysulfosuccinimide (sulfo-NHS) acetate blocking solution for 15 minutes; ii) Washing the biological sample with a reporter washing buffer, wherein the reporter washing buffer contains 0.5% Tween-20 in a 1× sodium chloride-sodium phosphate-EDTA (SSPE) solution; iii) Incubate the biological sample in an autofluorescence suppression buffer and / or irradiate the biological sample with blue light and / or ultraviolet (UV) light to quench the autofluorescence of the sample via photobleaching; and iv) Wash the biological sample with reporter washing buffer. The method according to any one of claims 1 to 9, further comprising the step of pre-treating a biological sample.

11. A method according to any one of claims 1 to 10, A) Step (a) comprises incubating the biological sample with a solution containing a reporter probe at a concentration of 5 nM in diethyl pyrocarbonate (DEPC) treated water, an 8.75 × SSPE solution, 0.5% Tween-20, and optionally 0.1% RNase inhibitor for at least 15 minutes. B) Step (b) comprises washing the biological sample with a reporter washing buffer, wherein the reporter washing buffer contains 0.5% Tween-20 in a 1× sodium chloride-sodium phosphate-EDTA (SSPE) solution, C) The step (c) is i) Immerse the biological sample in imaging buffer; and ii) Imaging biological samples to record the attributes and spatial arrangement of detectable labels of hybridized reporter probes. Including, and / or, D) The above step (d) is: i) Irradiating the biological sample with sufficient UV light to cleave the photocleavable linker portion in the hybridized reporter probe, and The washing of a biological sample with a strip washing buffer, wherein the strip washing buffer contains a 0.0033 × SSPE buffer and a 0.5% Tween-20 solution. This includes performing at least one or both of the following; and optionally, ii) Immersing a biological sample in an imaging buffer; and iii) further comprising imaging the sample to confirm that no detectable labels remain, The aforementioned method.

12. A method according to any one of claims 1 to 11, The method further includes performing a morphological scan of the biological sample to determine one or more regions of interest, Performing the aforementioned morphological scan: i) Staining the biological sample with a membrane-specific fluorescent stain, and then imaging the biological sample to identify the spatial arrangement of cell membranes within the sample; ii) Staining the biological sample with a nuclear-specific fluorescent stain and imaging the biological sample to identify the spatial arrangement of cell nuclei within the sample; and iii) Perform cell segmentation. The method according to any one of claims 1 to 11, further comprising at least one of the following.

13. Before step i) of contacting the biological sample with at least one nucleic acid probe, aa) A step of preparing a mounted biological sample by mounting the biological sample onto a functionalized microscope slide, wherein the biological sample is a formalin-fixed paraffin-embedded (FFPE) microtome section; bb) A step of firing the attached biological sample; cc) A step of deparaffinizing the attached biological sample; dd) A step of performing a targeted activation reaction on the attached biological sample; ee) A step of permeabilizing (clearing) the attached biological sample; ff) A step of applying at least one reference marker to the attached biological sample; and gg) The process of fixing the attached biological sample. The method according to any one of claims 1 to 12, wherein the biological sample is further prepared by means of the method.

14. The method according to any one of claims 1 to 13, further comprising assembling the fitted biological sample into a flow cell after step (ii).

15. The method according to claim 13, wherein the functionalized microscope slide is a microscope slide functionalized with (3-aminopropyl)trimethoxysilane (APTMS).

16. The method according to any one of claims 1 to 15, wherein the biological sample is an FFPE microtome section of a human tissue sample.

17. The method according to any one of claims 13 to 16, wherein step (bb) comprises firing the attached biological sample at 60°C for 1 hour.

18. The step (cc) is i) Incubate the attached biological sample in a xylene solution for 5 minutes; ii) Incubate the attached biological sample in the second xylene solution for 5 minutes; iii) Incubate the attached biological sample in the first 100% ethanol solution for 2 minutes; iv) Incubate the attached biological sample in a second 100% ethanol solution for 2 minutes; and v) Dry the attached biological sample at 60°C for 5 minutes. The method according to any one of claims 13 to 17, including the method described in any one of claims 13 to 17.

19. The above process (dd) is i) Incubate the attached biological sample at 100°C in a target activating solution containing Tris and EDTA solution with a pH of 9 for the time specified in Table 1; ii) Incubate the attached biological sample in DEPC-treated water for 15 seconds; iii) Incubate the attached biological sample in a 100% ethanol solution for 3 minutes; iv) Dry the attached biological sample. The method according to any one of claims 13 to 18, including the method described in any one of claims 13 to 18.

20. The above step (ee) is i) Incubate the attached biological sample in a proteinase solution at 40°C, wherein the proteinase solution contains protease K; ii) Wash the biological sample with the first aliquot of DEPC-treated water; and iii) Wash the biological sample with the DEPC-treated water from the second aliquot. The method according to any one of claims 13 to 19, including the method described in any one of claims 13 to 19.

21. The aforementioned process (ff) i) Incubating the attached biological sample for 5 minutes in a solution containing at least one reference marker, wherein the solution containing at least one reference marker is a solution containing carboxylated microspheres stained red, yellow, blue and / or green in a concentration of 0.0005% to 0.003% in 2 × saline-sodium citrate Tween (SSCT) solution; and ii) Wash the attached biological sample with 1x phosphate-buffered saline (PBS), The method according to any one of claims 13 to 20, comprising:

22. The above step (gg) is, i) Incubate the mounted biological sample in 10% neutral buffered formalin (NBF) for 1 minute; ii) Incubate the attached biological sample in the first Tris-glycine buffer solution for 5 minutes; iii) Incubate the attached biological sample in a second Tris-glycine buffer solution for 5 minutes; and iv) Incubate the attached biological sample in 1×PBS for 5 minutes. The method according to any one of claims 13 to 21, including the method described in any one of claims 13 to 21.

23. After the above step (gg), the attached biological sample is incubated in a blocking solution, and here the step of incubating the attached biological sample in a blocking solution is i) Incubating the mounted biological sample in a sulfo-NHS-acetic acid / Tween20 solution for 15 minutes, wherein the sulfo-NHS-acetic acid / Tween20 solution contains 100 mM sulfo-NHS-acetic acid and 0.5% Tween20 in 100 mM sodium phosphate pH 8; and ii) Incubating the attached biological sample in 1×PBS for 5 minutes. The method according to any one of claims 13 to 22, including the method described in any one of claims 13 to 22.

24. a) The step of incubating the attached biological sample with a solution containing multiple ISH probes is: The method includes incubating the biological sample with a solution containing multiple ISH probes at 37°C for 16 to 18 hours, thereby hybridizing at least one ISH probe to the target analyte in the biological sample, and / or b) The step of washing the biological sample is i) Incubate the mounted biological sample with the first 2×SSC solution; ii) Incubating the attached biological sample in the first formamide solution; iii) Incubating the attached biological sample in a second formamide solution; iv) Incubating the attached biological sample with a second 2×SSC solution; and, v) Incubate the attached biological sample with a third 2×SSC solution. including, The method according to any one of claims 1 to 23.

Images