Compositions and methods for spatial imaging applications

Abstract

This disclosure generally relates to spatial imaging methods and systems, as well as compositions and kits for use in such methods. Reagents and methods are provided herein that can be used for successful detection using fluorescent and bright-field microscopes and spectral imaging systems of a wide range of various protein markers across numerous types of biological samples.

Description

COMPOSITIONS AND METHODS FOR SPATIAL IMAGING APPLICATIONSFIELD

[0001] This disclosure generally relates to spatial imaging methods and systems, as well as compositions, kits, and systems for use in such methods.BACKGROUND

[0002] Spatialomics is an expanding research area focused on integrating spatial knowledge of tissue with transcriptomics (RNA) and proteomics (protein). Understanding the complexity of the biological structure(s) is an important biological process in cancer immunotherapy research which requires accurate target classification. Microscopy of sectioned tissue samples stained with fluorescent dyes and / or immunostaining (e.g., direct or indirect staining with primary or secondary antibodies conjugated to fluorophores) provides valuable histological, cellular and biomarker information. For example, translational profiling can be explored using multiple types of fluorescent dyes and / or immunostaining techniques. Fluorescence microscopy is commonly used to capture images of tissue stained with fluorophores (e.g., staining targets, dyes). Each fluorophore has a unique spectral emission profile spread over a plurality of fluorescence channels (e.g., wavelengths of light). Although each fluorophore has a unique spectral range and peak intensity, the spectral emission profile of multiple fluorophores may overlap. Accordingly, a spectral image acquisition operation may be performed to acquire both mixed and unmixed images of the sample. While the mixed image includes all fluorescent channels, the unmixed image includes only the desired fluorescent channels. However, concurrent interrogation of four or more targets on a single sample can be difficult in many ways due to the complexity involved with panel design, staining protocols, and data analysis. Designing a reliable multi-target biomarker panel requires consideration of the impact of protein abundance and localization, fluorophore compatibility, varying tissue types, and data characterization. Further, to address the needs of high target multiplex-ability (e.g., detection of 5 or more targets), cyclic detection labeling methods can facilitate iterative labeling and detection using automated fluidics.However, these approaches suffer from low simultaneous target throughput. Thus, there is a need for improved reagents and methods to facilitate multiplex imaging for spatial biology applications.SUMMARY

[0003] Compositions, methods, and systems described herein generally relate to capturing spectra using fluorescence microscopy. Reagents and methods are provided herein that can be used for successful detection using fluorescent microscopes and spectral imaging systems of a wide range of various protein markers across numerous tissue organs used in various biological research applications with a uniform technique labeling technique. Reagents, methods and systems provided herein can be used to analyze single cells in spatial context to provide a variety of information including cellular identity in time and space; cell types, cell states and cell functions; cell-cell interactions; cellular neighborhoods; and / or tissue microenvironments and architecture. Workflows provided herein can be used to interrogate immunology targets. Labeled samples and antibody-based detection approaches described herein can be employed to characterize how cells interact across complex tissues. Methods disclosed herein can be used with various types of imaging techniques and instrumentation. In certain embodiments, imaging can utilize a high-resolution spatial slide scanner, where a complete tissue section can be scanned in each of the relevant fluorescent channels. Such systems can be used to assemble a multichannel image that is suitable for spectral unmixing, review and data analysis.

[0004] Also provided herein are reagents and methods to facilitate multiplexed spatial phenotyping in tissue samples. Further, the described processes offer a streamlined approach for tissue labeling that can provide labeling with high plex panels and can be completed within several hours or less (e.g., less than about 2 hours).

[0005] In one aspect, a method for preparing a biological sample is disclosed that includes: a) providing a biological sample covalently bound to two or more populations of tyramides, each population including a plurality of tyramides, and each tyramide in the plurality of tyramides is linked to a detectable label, wherein the detectable labels in each population are the same, and wherein the detectable labels in different populations are unique; and b) removing non- covalently bound components from the biological sample.

[0006] In another aspect, a method for preparing a biological sample is disclosed that includes: a) providing a biological sample covalently bound to a population of tyramides, including a detectable label, wherein the detectable label is a fluorophore that emits light with an emissionmaximum in the far-red or near-IR spectral region upon excitation at an appropriate wavelength of light; and b) removing non-covalently bound components from the biological sample.

[0007] The described methods can further include covalently binding a first population of tyramides to the biological sample, the biological sample including a first marker, in a first HRP- mediated signal amplification reaction under a first set of conditions, wherein the first populations of tyramides binds in a localized area to the biological sample in the vicinity of the first marker, to provide a biological sample labeled covalently to a first population of detectable labels. The described methods can further include performing a first antigen retrieval step to unmask a second marker on the tissue sample.

[0008] In certain embodiments, the methods can further include: a) covalently binding a second population of tyramides to the biological sample, in a second HRP-mediated signal amplification reaction under the first set of conditions (e.g., temperature, reaction time, buffer, and hydrogen peroxide concentration), wherein the second population of tyramides binds in a localized area to the biological sample in the vicinity of the second marker, wherein each tyramide in the second population includes a second fluorophore, to provide a biological sample labeled covalently to a second population of fluorophore; and b) removing non-covalently bound components remaining from the second HRP-mediated signal amplification reaction from the biological sample.

[0009] In yet another aspect, a method of labeling a biological sample is disclosed that includes: a) providing a biological sample including a plurality of unique markers; b) treating the biological sample with multiple iterations of a labeling cycle, represented as [I]n, wherein l is a labeling cycle and n is the number of labeling cycles, wherein n = 2-20, wherein the labeling cycle (I) includes: i. covalently binding a plurality of populations of tyramides to the biological sample, in an HRP-mediated signal amplification reaction under a first set of conditions, wherein each population of tyramides binds in a localized area to the biological sample in the vicinity of a marker, wherein each tyramide in within a population comprises the same fluorophore, and wherein each population of tyramides includes a unique fluorophore, to provide a biological sample labeled covalently to a population of fluorophores; and ii. removing non-covalently bound components remaining from the HRP-mediated signal amplification reaction from the biological sample, to provide a biological sample labeled with multiple unique populations of fluorophores, wherein the number of unique populations of fluorophores corresponds to thenumber of labeling cycles (n). Each iteration can further include an antigen retrieval step prior to step (b)(i) to unmask the marker on the tissue sample.

[0010] An HRP-mediated signal amplification reaction can be employed in the instant methods that includes: (i) binding a primary antibody to a marker in the biological sample to provide a primary antibody treated sample; (ii) binding a secondary antibody, wherein the secondary antibody is attached to one or more horseradish peroxidase (HRP) molecules, to the primary antibody to provide an HRP treated sample; and (iii) treating the first treated sample with a detectable tyramide under conditions to initiate the signal amplification reaction.

[0011] For any of the methods disclosed herein, the method can further include detecting fluorescence emission from the populations of fluorophores. In certain embodiments, a first population of fluorophores emits light with an emission maximum greater than 710 nm and exhibits a first fluorescence signal intensity. For example, a first population of fluorophores can emit light with a first emission maximum between about 710 nm to about 850 nm. In certain embodiments, the method can utilize a second population of fluorophores that emits light with a second emission maximum between about 680 nm to about 720 nm and exhibits a second fluorescence intensity. In certain embodiments, the first fluorescence intensity and the second fluorescence intensity differ by a factor of 5 or less. The first population of tyramides can used at a first concentration in a first cycle, and a second population of tyramides can used at a second concentration in a second cycle, such that the first and second fluorescence intensities differ by a factor of 5 or less. Methods described herein can further include imaging the biological sample, wherein a first population of fluorophores and a second population of fluorophores are detected in two neighboring channels of a spectral imaging system, wherein the first population of fluorophores is detected in a first channel and the second population of fluorophores is detected in a second channel. For example, imaging the sample can be used to identify the locations the markers in the biological sample.

[0012] In yet another aspect, a biological sample is disclosed that includes two or more different markers, wherein each marker is labeled with two or more detectable tyramides, wherein each of the two or more detectable tyramides is covalently bound to the sample in a localized area to the biological sample in the vicinity of the two or more different markers, wherein each of the two or more detectable tyramides includes a different fluorophore, respectively, wherein a first fluorophore emits light with an emission maximum between 350 nmand 710 nm and a first fluorescent signal intensity upon excitation at an appropriate wavelength of light, and a second fluorophores emits light with an emission maximum between 710 nm and 850 nm and a second fluorescence signal intensity, wherein the first fluorescence intensity and the second fluorescence intensity differ by a factor of 5 or less. In certain embodiments, the biological sample is labeled with eight (8) or more unique detectable tyramides, including eight or more different fluorophores, respectively. A biological sample, as disclosed herein, can be disposed on an imaging support such as a microscope slide, cuvette, well or dish and can be embedded in a mounting medium, wherein the mounting medium has a refractive index of 1.47 - 1.52.

[0013] In yet another aspect, a composition is provided that includes a) a first population of tyramides at a first concentration, wherein the first population includes a plurality of first fluorophores capable of emitting light with an emission maximum between 350 nm and 710 nm and a first fluorescent signal intensity; and b) second population of tyramides at a second concentration, wherein the second population includes a plurality of second fluorophores capable of emitting light with an emission maximum between 710 nm and 850 nm and a second fluorescence signal intensity, wherein the first and second fluorescence intensity differ by a factor of 5 or less. In some embodiments, the concentration of the first population of tyramides is different from the concentration of the second population of tyramides.

[0014] In yet another aspect, a kit for labeling a biological sample is disclosed that includes a) two or more populations of tyramides, wherein each population of tyramides includes a plurality of unique fluorophores capable of emitting light with an emission maximum between about 350 nm and about 850 nm upon excitation at an appropriate wavelength of light, wherein at least one population of fluorophores emits light with an emission maximum in the far-red or near-IR spectral region; a poly-HRP- labeled detection reagent; an antigen retrieval reagent; and instructions for performing two or more cycles of HRP-mediation signal amplification to label the tissue sample with the two or more populations of tyramides.

[0015] In yet another aspect, an imaging system that includes an imaging device and a biological sample, as disclosed herein, mounted in the imaging device. In certain embodiments, the imaging system can include an imaging device, and a controller. The controller can include an electronic processor and a non-transitory, computer readable medium, wherein the controller is configured to receive a selection of one or more fluorescent channels for imaging a biologicalsample, as disclosed herein; capture, with the imaging device, a raw image of the sample; and unmixing the raw image to generate an unmixed image.

[0016] In any of the compositions, samples, kits, systems, or methods disclosed herein, the biological sample can be a tissue, cell, cell organoid, cell spheroid, 3D cell culture, or a whole organism; and the detectable label can be a fluorophore or chromogen or a combination thereof, such as a cyanine-based dye, a hemi-cyanine-based dye, a rhodamine-based dye, a coumarin- based dye, a pyrene-based dye, an indacene-based dye (e.g., BODIPY), or an indole-based dye (e.g., DAPI (4',6-diamidino-2-phenylindole), and the detectable label can further include a watersolubilizing group, such as a polyethylene glycol) or sulfonate group..BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 is a general schematic for a spatial biology workflow.

[0018] FIG. 2 is a schematic of a multiplex labeling workflow depicting covalent attachment of a labeling reagent.

[0019] FIG. 3 illustrates an exemplary labeling workflow to interrogate 8 markers having low, medium, and high abundance using a combination of 4 rounds of enzymatic labeling, followed by labeling with an antibody mix / cocktail.

[0020] FIG. 4 shows (A) an 20X image of FFPE human tonsil labeled with a set of 8 fluorescent tyramides that emit between 430 nm and 750 nm and DAPI nuclear counterstain collected on an EVOS SI 000 spectral imaging system with spectral unmixing. Images of each dye are shown in an individual channel (B-J).

[0021] FIG. 5 shows set of images of FFPE human duodenum tissue acquired on an EVOS M7000 system with individual filters for each of the dyes. (A) is an image to illustrate three different primary antibody detection strategies that can be multiplexed together on a single sample. (B) is an image showing that a single type of primary antibody can be multiplexed together on a single sample, in this case, mouse primaries were detected using the mouse secondary HRP. (C) is an image showing an alternative multiplex staining strategy.

[0022] FIG. 6 is a general staining workflow for IHC depicting labeling using a primary antibody conjugate mixture with an organic fluorophore.

[0023] FIG. 7 shows a set of images of tissues labeled with primary antibody conjugates. Primary antibody conjugates were confirmed and evaluated against secondary antibody IHClabeling. Primary antibody conjugates were specific and comparable to primary-secondary for the three different targets. (A) PanCK (cytokeratin), epithelial cells labeled on normal human tonsil with Alexa Fluor™ 514; (B) CD68, macrophage marker labeled on normal human tonsil with Alexa Fluor™ 488 Plus; (C) FoxP3, transcriptional regulator labeled on normal human tonsil with Alexa Fluor™ Plus 647.

[0024] FIG. 8 is an image of human tonsil tissue labeled with primary antibody conjugates against PanCK (AE1 / AE3) Alexa Fluor™ 700 (seen in red), CD20 (L26) Alexa Fluor™ Plus 750 (seen in purple), CD68 (KPI) Alexa Fluor™ 488 (seen in blue) and Ki67 (SolA15) eFluor™ 506 (seen in green).

[0025] FIG 9 is a set of images of FFPE human colon adenocarcinoma labeled using primary IHC validated antibody conjugates. (A) Composite image of tissue labeled with multiple labels and images for individual channels: (B) PanCK (AE1 / AE3) eFluor™ 506 (seen in green); (C) Ki67 (SolA15) Alexa Fluor™ 514 (seen in yellow); (D) CD8a (C8 / 144B) Alexa Fluor™ 594 (seen in red); and (E) SMA (1 A4) Alexa Fluor™ 700 (seen in blue).

[0026] FIG. 10 is an image of FFPE human small intestine labeled with four dyes to demonstrate individual target validation.

[0027] FIG. 11 is a set of images illustrating the contrasting cellular phenotypes between heathy and diseased tissue. (A) normal human tonsil and (B) Non-Hodgkin lymphoma human tonsil.

[0028] FIG. 12 is a set of images of normal human tonsil tissue labeled with primary antibody conjugates collected using methods disclosed herein. (A) DAPI nuclear counterstain; (B) Ki67, proliferative marker labeled on normal human tonsil tissue with eFluor™ 506; (C), CD20, B-cell marker labeled on normal human tonsil tissue with DY 396XL (Dyomics); (D) CD31, endothelial cell marker labeled on normal human tonsil tissue with Alexa Fluor™ 488; (E) CD8a, T-cell marker labeled on normal human tonsil tissue with Alexa Fluor™ 514; (F) Pan-CK (cytokeratin), endothelial cells labeled on normal human tonsil tissue with DY-51 IXL (Dyomics); (G); CD38, cyclic ADP ribose hydrolase positive cell marker labeled on normal human tonsil tissue with Atto490LS (H) CD4, T-cell marker labeled on normal human tonsil tissue with Alexa Fluor™ 555; (I) CD68, macrophage marker labeled on normal human tonsil tissue with Alexa Fluor™ 568; (J) CD45RB, memory T-cell marker labeled on normal human tonsil tissue with Alexa Fluor™ 610; (K) PCNA, nuclear marker that indicates cell proliferationlabeled on normal human tonsil tissue with DY-521 XL (Dyomics GmbH); (L) PD-L1, programmed cell death ligand- 1 marker labeled on normal human tonsil tissue with Alexa Fluor™ 647; (M) CD3d / e, pan-T-cell marker labeled on normal human tonsil tissue with Alexa Fluor™ 660; (N) E-cadherin, epithelial cell marker labeled on normal human tonsil tissue with Alexa Fluor™ 700; (O) HLA-DR, effector T-cell marker labeled on normal human tonsil tissue with DY-731 (Dyomics); (P) PD-1, programmed cell-death-1 T-cell marker labeled on normal human tonsil tissue with Alexa Fluor™ 750; (Q) Vimentin, structural integrity cells labeled on normal human tonsil tissue with DY-805 (Dyomics); and (R) SMA, Smooth Muscle Actin marker labeled on normal human tonsil tissue with DY-831 (Dyomics).

[0029] FIG. 13A is a general spatial biology imaging workflow.

[0030] FIG. 13B is a diagram illustrating a cyclic labeling process for multiplex labeling of a tissue sample, as described herein.

[0031] FIG. 14 shows a set of spectral images showing differences in FFPE human tonsil tissue types that has been labeled using IHC validated primary antibody dye conjugates.

[0032] FIG. 15A show an image of an 81mm2area of FFPE invasive ductal carcinoma collected on the EVOS S1000 Spatial Imaging System with spectral unmixing. The inset shows zoomed in portion to highlight the details of the stained image. FIG. 15B shows images, as shown in the inset, for individual unmixed channels for tissue sample labeled using 9 fluorescent labels with emission maxima ranging from 430 nm to 750 nm and primary antibodies raised against tissue targets: DAPI nuclear counterstain (A), Dye 430 with Vimentin (B); Dye 488 with CD8 (C); Dye 514 with CD68 (D); Dye 555 with PCNA (E); Dye 594 CD4 (F); Dye 647 with Prohibitin (G); Dye 700 with CD3 (H); and Dye 750 with CD20 (I).

[0033] FIG. 16 shows a set of images demonstrating the analysis of the tissue sample region in FIG.15 (B) stained with 8 dyes and DAPI nuclear counterstain. (A) 0.7 mm2area analyzed containing 10454 cells, with magenta segments indicating B cells, blue segments indicating macrophages, green segments indicating cytotoxic T cells, yellow segments indicating helper T cells, orange segments indicating proliferating cells, grey segments indicating cells that did not classify into any of the identified phenotypes, and white cell segments indicating more than one positive proliferating & immune cell phenotypes. (B) Tissue segmentation identifying vimentin positive and negative regions. (C) Identification of region-specific cell localization. Cellular phenotyping enabled characterization of the 1.06 million cells identified in the 81mm2tissuesection shown in (A). 25% of the cells were assigned as immune cells, and 23% of the nonimmune cells were proliferating.

[0034] FIG. 17 shows analysis plots derived from the imaging data from FIG. 16.

[0035] FIG. 18 shows images of two FFPE human intestine tissue samples collected using EVOS M7000 microscope. Both samples were imaged at match exposure conditions. The left panel (A) shows an image after staining of smooth muscle actin (SMA) on FFPE human intestine with Aluora™ 750 dye (Thermo Fisher Scientific). The right panel (B) shows a spectral image after staining of SMA on FFPE human intestine, where the sample was subject to 7 stripping cycles and then stained with the Aluora 750 dye.

[0036] Additional embodiments and advantages will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0037] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.

[0038] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (several) embodiment(s) and together with the description serve to explain the principles described herein.DETAILED DESCRIPTION

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of microscopy, biochemistry, molecular biology, immunology, microbiology, genetics, cell and tissue culture, and protein and nucleic acid chemistry described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.

[0040] As used herein, terms such as “include,” “including,” “contain,” “containing,” “having,” and the like mean “comprising.” The present disclosure also contemplates otherembodiments “comprising,” “consisting essentially of,” and “consisting of’ the embodiments or elements presented herein, whether explicitly set forth or not. As used herein, “comprising,” is an “open-ended” term that does not exclude additional, unrecited elements or method steps. As used herein, “consisting essentially of’ limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. As used herein, “consisting of’ excludes any element, step, or ingredient not specified in the claim.

[0041] As used herein, the term “a,” “an,” “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. In addition, “a,” “an,” or “the” means “one or more” unless otherwise specified. As used herein, the term “or” can be conjunctive or disjunctive. As used herein, the term “and / or” refers to both the conjunctive and disjunctive. As used herein, the term “substantially” means to a great or significant extent, but not completely.

[0042] As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In one aspect, the term “about” refers to any values, including both integers and fractional components that are within a variation of up to ± 10% of the value modified by the term “about.” Alternatively, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, in some embodiments within 5 -fold, and in some embodiments within 2-fold, of a value. As used herein, the symbol means “about” or “approximately.”

[0043] All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1, 0.2, 0.3, 0.4 . . . 2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to ±10% of any value within the range or within 3 or more standard deviations, including the end points, or as described above in the definition of “about.”

[0044] “Antibody” as used herein means an immunoglobulin or a fragment thereof and encompasses any polypeptide including an antigen-binding site regardless of the source, method of production, and other characteristics.

[0045] An “analyte” or “antigen” as used herein refers to any substance recognized by an antibody, or another means of detection.

[0046] A “biological sample” or “biological specimen” as used herein encompasses hematological, cytological and histological specimens, such as cells, 3D cell cultures (e.g. spheroids and organoids), whole organisms (e.g. flies, worms, zebrafish), cell-free extracts, or a fluid sample (e.g., blood, sputum, urine, saliva, and the like), a tissue (e.g., whole tissue or tissue fraction), a tissue mimic (e.g., synthetic tissue), a cell (e.g., a cell from a mammal, yeast, fungi, or bacteria), a cell mimic (e g., synthetic cell), a cell culture (e.g., a 2D or 3D cell culture sample), a cell organoid, a cell spheroid, a whole organism, and the like. A tissue sample can be any type of nervous, epithelial, muscular, and connective tissue, including an organ tissue. Biological samples can be from a plant or animal (e.g., human, mouse, fly, worm, fish, frog, fungi, and the like).

[0047] A “conjugate” or “conjugated,” as used herein refers to two or more moieties directly or indirectly coupled together. For example, a first moiety may be directly covalently linked to a second moiety. Indirect attachment is possible, such as by using a "linker" (a molecule or group of atoms positioned between two moieties). Other examples of detectable labels and substances that can be conjugated (i.e., linked) to detectable labels include polymers, polymer particles, bead or other solid surfaces and substrates.

[0048] A “detectable label” as used herein refers to any molecule which may be detected directly or indirectly to reveal the presence of a target in the sample. A detectable label can be a molecule or material that can produce a detectable (such as visually, electronically or otherwise) signal that indicates the presence and / or concentration of a target, such as a target molecule, in a sample, such as a tissue sample. A direct detectable label can be detected without the need for additional molecules. When conjugated to a specific binding molecule, the detectable label can be used to locate and / or quantify the target to which the specific binding molecule is directed. Thereby, the presence and / or concentration of the target in a sample can be detected by detecting the signal produced by the detectable label. A detectable label can be detected directly or indirectly, and several different detectable labels conjugated to different specific -binding molecules can be used in combination to detect one or more targets. Multiple detectable labels that can be separately detected can be conjugated to differentspecific binding molecules that specifically bind different targets to provide a multiplexed assay that can provide detection of the multiple targets in a sample.

[0049] Examples of detectable labels that can be used herein include, but are not limited to, fluorophores, chromophores, chemiluminescent compounds (e.g., luminol, isoluminol, acridinium esters, 1,2-dioxetanes, and pyridopyridazines), electrochemiluminescent labels (e.g., ruthenium derivatives), bioluminescent labels, and enzymes that catalyze a color change in a substrate. For some detectable labels, the target is detected by the presence of a color, or a change in color in the sample. More than one type of color may be used, for instance, by attaching distinguishable labels to a single detection unit or by using more than one detection unit, each carrying a different and distinguishable label. Other examples of detectable labels that can be used in spatial biology workflows, either alone or in combination with the methods described herein include gold or other metal particles, heavy atoms, spin labels, radioactive substances such as radioisotopes, and quantum dots.

[0050] Indirect detectable labels may be used, which require the employment of one or more additional molecules. Examples include enzymes that affect a color change in a suitable substrate, as well as any molecule that may be specifically recognized by another substance carrying a label or react with a substance carrying a label. Other examples of indirect detectable labels thus include antibodies, antigens, nucleic acids and nucleic acid analogs, ligands, substrates, and haptens. Detectable labels can be attached (e.g., conjugated) to a variety of different substances, including, without limitation, haptens, antigens, nucleic acids or nucleic acid analogues, proteins, such as receptors, peptide ligands, enzymes, enzyme substrates, or antibodies (including antibody fragments).

[0051] “Fluorophore” as used herein is a molecule that emits detectable electromagnetic radiation upon excitation with electro-magnetic radiation at one or more wavelengths. A large variety of fluorophores are known in the art and are developed by chemists for use as detectable molecular labels and can be conjugated to affinity molecules described herein. The term “fluorophore” and “fluorescent dye” may be used interchangeably herein.

[0052] “Chromogen” as used herein refers to a chemical compound that can, by chemical or other means, be converted into a chromophore. The term “chromophore,” as used herein, refers to an aromatic compound including a chemical grouping that gives color to the compound by causing displacement of, or appearance of, absorbent bands in the visible spectrum. Exemplarychromophores include, but are not limited to, R-N=N- (e ., azo dyes). Exemplary chromogens include, but are not limited to, naphthols, aryl diazonium salts, and 1,3 -diketones. Examples of chromogens include 3,3'-diaminobenzidine tetrahydrochloride (DAB) and the naphthol tyramide conjugates described in WO2024 / 145053A1, incorporated herein by reference in its entirety.

[0053] The terms “recognize,” “recognition,” or “recognizing,” etc., as used herein, mean an event in which one substance, such as an affinity molecule, directly or indirectly interacts with a target in any way such that the interaction with the target may be detected by an affinity molecule. In some nonlimiting examples, a probe may react with a target, or directly bind to a target, or indirectly react with or bind to a target by directly binding to another substance that in turn directly binds to or reacts with a target.

[0054] “ Target,” also used interchangeably with “analyte,” as used herein refers to any substance present in a sample that is capable of being detected. A target or analyte can include a protein, such as a glycoprotein or lipoprotein, phosphoprotein, methylated protein, or a protein fragment, a peptide, or a polypeptide. A target or analyte can include a nucleic acid segment or a nucleic acid analog segment. A target or analyte can be an antigen or an antibody. A target or analyte can include one or more of lipids; glyco-lipids; carbohydrates; polysaccharides; salts; ions; or a variety of other organic and inorganic substances. A target or analyte can be expressed on the surface of the sample, such as on a membrane or interface. Alternatively, a target or analyte can be contained in the interior of the sample. In the case of a cell sample, for instance, an interior target or analyte can include a target or analyte located within the cell membrane, periplasmic space, cytoplasm, or nucleus, or within an intracellular compartment or organelle. A target or analyte can also include viral particles, or portions thereof, such as nucleic acids or proteins. The viral particle can be a free viral particle, i.e., not associated with any other molecule, or it can be associated with any sample described above.

[0055] The term “multiplex” or “multiplexing” refers to the ability to analyze and visualize two or more molecular markers or data types. Within the context of the spatial biology field, multiplex detection refers typically refers to detection of more than five molecular markers or data types within the same biological sample (e.g., tissue section or cell), while preserving spatial information. “Multiplexing” also refers to techniques for measuring the expression or presenceof multiple genes, proteins, or other molecules in the same tissue sample, while maintaining their spatial location.

[0056] As used herein, “tyramide” or “detectable tyramide” refers to a traditional tyramine- containing substrate in which a tyramine group is linked to a detectable label such as a fluorophore, chromogen, or hapten, and that can become activated in the presence of a peroxidase. “Tyramide,” as used herein, also refers to phenol-containing tyramide-like derivatives and analogues, such as the iFluor Styramide™ reagents from AAT Bioquest (Pleasonton, CA), that can participate in HRP -mediated amplification workflows that enhance detectable signal for low abundance targets in tissues or cells. Tyramides that are conjugated (e.g., covalently bonded directly or indirectly through a linker) to a detectable label, as disclosed herein, can bind covalently to exposed tyrosine groups of proteins in or a biological substance, such as a tissue, when utilized in enzyme-catalyzed amplification workflows to provide a tissue that is labeled covalently to a detectable label, such as a fluorophore, chromogen, or hapten (e.g., biotin or DIG).

[0057] Described herein are methods for staining multiple targets (e.g., immunologically relevant markers) in a biological sample (e.g., whole tissue sections or cells) with spectrally unique detectable labels. Microscopy of biological samples (e.g., sectioned tissue) stained with detectable labels (e.g., fluorescent dyes) and / or immunostaining (e.g., direct or indirect staining with primary or secondary antibodies conjugated to fluorophores) can provide valuable histological, cellular and biomarker information. Stained biological samples, prepared using methods described herein, can be visualized with advanced spectral imaging systems equipped with spectrally unmixed channels necessary for downstream analysis. Spectral imaging systems can produce high-resolution images and / or can increase flexibility in IHC detection platforms. Systems and methods are provided that can be used for detecting low-abundance targets in multiplex immunohistochemistry (mIHC) applications and can overcome known limitations to mIHC, spatial proteomics and other multiplex imaging experiments, especially when detecting low-abundance targets (e.g., proteins) over autofluorescence and non-specific background.

[0058] A general schematic for a spatial biology workflow that implements labeling techniques is depicted in FIG. 1. An FFPE tissue sample is first mounted on a slide. The mounted tissue sample then can undergo dewaxing and retrieval steps. The tissue sample then can be labeled. The label can be a fluorescently-labeled antibody conjugate. The antibodyconjugate can be an IHC validated primary conjugate. Alternatively, or in addition, an antibody prepared using a commercial antibody labeling kit, such as the ReadyLabel™ Antibody Labeling Kits (available from Thermo Fisher Scientific) or using an enzyme-mediated amplification technique. Once labeled, the tissue sample can be mounted and imaged using a spectral imaging system. The image data collected by the system can undergo a spectral unmixing process, and the unmixed spectral image data can be reviewed and analyzed.

[0059] FIG. 13A shows a general spatial biology imaging workflow for tissue staining that can be used to image immunology targets labeled with antibody-based detection. Immunology targets labeled with antibody-based detection can be used to characterize how cells interact across complex tissues imaged on a high-resolution spatial slide scanner. The workflow includes tissue staining (Step 1), image acquisition and exploration (Step 2), and image and data analysis (Step 3). In Step 1 of a typical workflow, an FFPE embedded tissue sample is mounted on an imaging support (e.g., a microscope slide), dewaxed and subjected to antigen retrieval, and then labeled with a detectable label. Samples are prepared to account for tissue type, tissue preservation components, tissue thickness, antigen localization, antigen abundance and autofluorescence characteristics. In Step 2 of workflow, images of the labeled sample are acquired, pre-processed (i.e., performing unmixing and stitching processes) and visualized. Labeled samples can be visualized, for example, using a high-speed, high-resolution imaging system, such as the EVOS S1000 Spatial Imaging System (Thermo Fisher Scientific). Once an image has been produced, features are extracted and further analyzed (Step 3) using analysis software, such as the Indica Labs HALO analysis software.

[0060] When multiple fluorescent reagents are used simultaneously for the same tissue sample spectral bleed-through, i.e., detection of fluorescence from neighboring fluorescent channels in the channel of interest, can hamper identification of actual targets. One workaround is to limit the number of targets by staining samples with a few spectrally separate fluorophores. However, multiplexing the detection is often required since the availability of tissue samples can be limited, and the tissue and the detection reagents can be expensive.

[0061] Spectral imaging systems can extract spectra of multiple fluorescent reagents to produce high quality individual images, where images of each fluorescent marker can be detected in a separate channel. Fluorescence imaging is based on photochemistry of dyes. To detect a fluorescence image with a camera, the chemical reactions initiated by light absorption,where molecules absorb photons, become excited, and then emit light (fluorescence) as they return to their ground state need to be studied. The fluorescence absorption spectrum and the fluorescence emission spectrum of a set of dyes needs to be known in to define dyes candidates for spectral unmixing at given plex. Based on that knowledge spectral characteristics of light source and filters are optimized. Precise knowledge of the signatures of dye fluorescence absorption (fluorescence excitation spectrum), and the dye emission (fluorescence emission spectrum) is needed to achieve successful unmixing at high plex. Although it can be advantageous to utilize imaging systems equipped with unmixing capabilities, especially when labeling with greater than 5 fluorophores. It is also contemplated that spectrally unique detectable labels can be interrogated using conventional imaging equipment that does not implement specialized spectral unmixing procedures.

[0062] In general, when acquiring data from biological samples with multiple overlapping fluorophores, it is desirable to separate the fluorophores into individual fluorophore channels. Accordingly, systems and methods have been devised for calibrating a fluorescence microscope to separate fluorophores within captured images. Calibration can include capturing an unstained image of a sample and a plurality of images of samples stained with a single fluorophore (referred to herein as single color control samples). The unstained image and the plurality of single color control sample images can be used to generate an unmixing matrix for desired fluorescence channels. The unmixing matrix is then used to extract individual fluorophore channels from multi-channel images of samples.

[0063] Fluorescent microscope calibration can significantly enhance the unmixing of individual channels for each fluorophore. By ensuring precise alignment and accurate calibration of the microscope, these techniques minimize spectral overlap and improve the distinction between different fluorescent signals. This results in clearer, more accurate imaging, allowing for better identification and analysis of the various fluorophores present in the sample. Consequently, the overall quality and reliability of fluorescence microscopy data are greatly improved.

[0064] Methods for spectral unmixing of imaged fluorophores are described in co-pending PCT application number, PCT / US2025 / 022946, filed April 3, 2025, entitled “Spectral Unmixing of Imaged Fluorophores,” incorporated herein by reference in its entirety. The method for generating a spectral profile can include receiving, by a computing device, one or more single-color control images, a foreground channel, and a background channel. As used in this disclosure, a foreground channel refers to a specific channel which includes the area or signal of interest. A background channel represents a surrounding area with minimal to no fluorescence from a dye which is used to estimate and subtract background noise which may include autofluorescence from the foreground signal. As used herein, autofluorescence is the natural fluorescent emission from biological structures or other substances in the presence of excitation light. The method includes determining, by the computing device, a difference between the foreground channel and the background channel, acquiring, by the computing device, a foreground pixel mask from the difference, averaging, by the computing device, a set of foreground pixels in the foreground pixel mask to generate a first spectrum, and subtracting, by the computing device, an unstained spectrum from the first spectrum to generate a second spectrum, wherein the second spectrum defines a spectral profile of the sample. As used in this specification, a pixel mask may be defined as the pixels from the foreground channel used in averaging the intensity in all the channels.

[0065] Also provided is a method of calibrating an imaging device. The method includes obtaining a first image of an unstained version of a sample, acquiring an unstained spectral profile of the sample using the first image, obtaining, for each of a plurality of stained versions of the sample, a second image, and extracting a plurality of spectral profiles associated with the plurality of stained images, each spectral profile associated a fluorophore of the plurality of fluorophores. In each of the stained images, a portion of the sample is stained with a different fluorophore of a plurality of fluorophores. The method includes generating an unmixing matrix based on the unstained spectral profile and the plurality of spectral profiles.

[0066] The imaging system can include a calibrated imaging device and a controller including an electronic processor and a non-transitory, computer readable medium. The controller is configured to receive a selection of one or more fluorescent channels for imaging a sample, where the sample is stained with a plurality of fluorophores, and capture, with the imaging device, a raw image of the sample. The controller is configured to apply an unmixing matrix to the raw image to generate an unmixed image and generate the unmixed image. The unmixing matrix can be generated by repeating image capture (e.g., by repeating a single color control (SCC) operation), where a single operation includes subtracting the background channel from the foreground channel; defining a pixel mask; and applying the pixel mask to all of the channels, ona plurality of calibration samples, each calibration sample in the plurality of calibration samples stained with a different fluorophore included in the plurality of fluorophores.

[0067] An exemplary spectral unmixing method is performed by a computing device that includes receiving one or more single-color control images, a foreground channel, and a background channel. The single-color control images are images of the sample with a single fluorophore applied to the sample, and the foreground channel and the background channel are associated with the fluorophore. The method can include determining a difference between the foreground channel and the background channel, acquiring a foreground pixel mask from the difference, averaging a set of foreground pixels in the foreground pixel mask to generate a first spectrum, and subtracting an unstained spectrum from the first spectrum to generate a second spectrum. The second spectrum defines a spectral profile of the sample.

[0068] Spatial proteomic workflows described herein solve various problems associated with prior approaches for multiplex labeling (i.e., detection of multiple targets) and imaging of biological samples. In spectral unmixing workflows, several limitations can be encountered. 1) Highly variable staining signal intensities between the different fluorophores can decrease ability to resolve the dimmer signals and requires a higher dynamic range of the acquisition instrumentation. This can result in signal levels in dimmer labels being obscured by noise from higher intensity ones, resulting in loss of performance. 2) Low levels of staining signal differentiation from background autofluorescence of the tissue. This can result in inability of unmixing algorithms to separate signal from autofluorescence, and result in loss of signal fidelity. 3) The need to label multiple targets using antibodies from the same host species, a challenge when relying solely on the specificity of secondary antibodies as employed in classic ICC / IHC workflows. This can result in limited flexibility of labeling and significantly hamper time to results. The workflows described herein solve one or more of these issues by employing a set of certain amplification modalities in combination with certain secondary antibody conjugates and optimized labeling conditions, to improve the overall signal of staining, without sacrificing signal to background. These reagent optimizations are designed to normalize the staining signal intensity across all fluorescent channels of the imaging system and provide a significantly higher signal intensity compared to tissue autofluorescence background.

[0069] The methods described herein can be used in an immunohistochemistry assay, an immunocytochemistry assay, an in-situ hybridization (ISH) assay, enzyme immuno-assays(EIA), enzyme linked immuno-assays (ELISA), blotting methods (e.g., Western, Southern, and Northern), labeling inside electrophoresis systems or on surfaces or arrays, or other general detection assays known in the art. As used herein, “Immunohistochemistry” or “IHC” refers to a technique that uses an antibody to bind a specific antigen in a tissue section and is visualized (e.g., imaged) with a fluorophore or colored substrate. Locations of stained proteins help the researcher understand cell types and their functions within a tissue. For example, immunohistochemical (IHC) staining provides a method of detecting targets in a sample or tissue specimen in situ. The overall cellular integrity of the sample is maintained in IHC, thus allowing detection of both the presence and location of the targets of interest. Typically, a sample is fixed with formalin, embedded in paraffin, and cut into sections for staining and subsequent inspection by light microscopy. Current methods of IHC use either direct labeling or secondary antibodybased or hapten-based labeling. Examples of known IHC systems include, but are not limited to, EnVision™ (DakoCytomation), Powervision® (Immunovision, Springdale, AZ), the NBA™ kit (Zymed Laboratories Inc., South San Francisco, CA), HistoFine® (Nichirei Corp, Tokyo, Japan).

[0070] Basic IHC staining typically requires sample preparation, antigen retrieval, blocking, target detection, and visualization. Traditional immunohistochemistry (IHC) approaches have been limited to resolving spectrally distinct signals for up to four (4) targets. More recently, spectral imaging systems equipped with spectral unmixing capabilities have become an increasingly important driver of multidimensional analysis of complex cell-tissue systems. For example, increasing the number of unique targets that can be detected in a single sample (e.g., greater than 4 targets) can be achieved through the unmixing of overlapping spectral signatures.

[0071] For spatial imaging applications, even with the application of spectral unmixing processes, fraction bleed through between channels can persist. “Fraction bleed through,” as used herein, refers to the ratio of the signal observed in off target channels of a spectral imaging system relative to the signal of the target fluorophore after unmixing has occurred. Fraction bleed through can be reduced by selecting fluorophores that exhibit discrete (i.e., well -separated) excitation and emission profdes with minimal spectral overlap.

[0072] Provided herein are compositions and methods to achieve multiplex spatial biology imaging of at least two populations of detectable labels. Each population possesses one or more spectral properties (e.g., excitation or emission maximum, fluorescence intensity, and the like) that can help minimize fraction bleed. A population (also referred to herein as a “set”) includes aplurality of unique detectable labels, as disclosed herein, such that the population can be detected by a spectral imaging system. For example, a population of detectable labels suitable for multiplex imaging can be a population of fluorophores with an excitation and / or emission profile within the UV / Visible to near-IR spectral region. For multiplex labeling with fluorophores, it is generally desirable that the excitation and / or emission spectral profiles do not overlap. For example, fluorophores with suitable spectral profiles can exhibit emission and / or excitation maxima that are spectrally resolved (i.e., the excitation and / or emission maxima do not overlap). For example, improved imaging resolution that the difference in excitation or emission maxima for a pair of fluorophores is desirably 10 nm or greater (e.g., 10 nm - 80 nm).

[0073] In some embodiments, a set of fluorophores for use in a multiplex spatial imaging assay emits light at a wavelength within the UV / Visible to the near-IR portion of the electromagnetic spectrum. For detection by spectral imaging systems currently on the market, detectable fluorophores typically emit light from about 350 nm to about 850 nm upon excitation at an appropriate wavelength of light (e.g., by laser excitation). In certain embodiments, a population of fluorophores can emit in the visible spectral region (e.g., 400 nm to 800 nm). In certain embodiments, the set of fluorophores can emit light in the far-red to near-IR spectral region (e.g., about 700 nm to about 850 nm). In certain embodiments, two or more sets of fluorophores can be implemented in a spatial imaging assay, where one set emits light in the visible spectral region (e.g., 400 nm to 800 nm) and a second set emits light in the far-red to near-IR spectral region (e.g., about 700 nm to about 850 nm).

[0074] A set of fluorophores for multiplex spectral imaging applications can include fluorophores that fall within the channels of the spectral imaging system. For the EVOS S1000 spectral imaging system, a channel refers to the combination of the light source and the filter set, whereas for traditional imaging systems, such as the EVOS M7000, a channel refers to the light cube. Although not required, it can be preferable that the fluorophore exhibits an emission maximum that falls with the range of a detector channel of the system. An exemplary fluorophore set for multiplex spectral imaging can include fluorophores that fall within the specified filter or light cube of the imaging system, as shown in Table 1. Multiplex fluorophore sets can be constructed using two or more of the dyes listed in Table 1. In certain embodiments, the multiplex fluorophore set includes 3, 4, 5, 6, 7, or 8 dyes from those listed in Table 1.Table 1: Spectral Properties for Multiplex Fluorophores

[0075] Suitable antibody panels can include fluorescent dyes that are sufficiently bright to overcome limitations associated with autofluorescence and non-specific background. Bright fluorophores can exhibit fluorescent signal intensity that is about two times or greater above detected background signal. While the ratio of positive fluorescent signal above the background fluorescent signal (i.e., signal -to-noise ratio) should be above 2 to correctly identify labeled targets, bright signals with a ratio of above 3 overcome many known limitations of mIHC spatial proteomics. Use of detectable labels that can generate high signal intensity, without added background can facilitate better spectral unmixing of the stained tissue. A higher signal intensity can enable higher signal of detection after applying linear unmixing algorithms, at least 2000 RFU, for more effective separation of target signal from non-stained regions and other stains on the tissue being studied. Due to the improved fluorescence sensitivity achieved by implementing spatial fluorophores that are exceptionally bright, it is possible to reduce primary antibody dilutions to 1: 1,000 to 1:50,000, in addition to achieving detection of extremely low-abundance targets over background noise when used in HRP enzyme-mediated signal amplification workflows.

[0076] Methods are disclosed herein for detecting low-abundance targets in multiplex IHC and spatial imaging applications. Certain methods use enzyme-mediated signal amplification to covalently label a biological sample (e g., tissue or cell) with detectable labels (e.g., fluorophores or chromogens). The covalent labeling methods have various advantages over non-covalent labeling methods used in standard IHC procedures. As demonstrated in the examples disclosed herein, bright fluorescent dyes have been implemented in enzyme-mediated signal amplificationmethods to label tissue samples with exceptionally bright, covalently attached fluorophores. The methods achieve localized deposition of the activated tyramides in the area on the sample surrounding the target (e.g., antigen) due to minimal diffusion from the site of enzyme activity. As a result, images produced using the labeled sample exhibit improved spatial resolution as compared to samples produced using other methods that do not achieve covalent attachment of detectable tyramides to the sample.

[0077] An exemplary tissue staining workflow (130) is depicted in FIG. 13B. In a first step, an FFPE embedded tissue sample (140) that includes an epitope (142) is mounted on an imaging slide (e.g., a microscope slide) (144). The tissue sample can be subjected to an antigen retrieval step to expose the target marker and mounted on the slide in a suitable mounting medium, such as the glycerol-based, hard-setting ProLong™ Glass Antifade Mountant from Thermo Fisher Scientific (Step 1). An exemplary antigen retrieval step can include heat-induced epitope retrieval (HIER) using either citrate buffer (pH 6.0) or EDTA (pH 9) using a microwave or pressure cooker according to standard antigen retrieval protocols. In Step 2, non-specific epitopes (146) of the marker on the tissue are blocked. The blocking step can be performed using methods commonly used in the art. For example, the blocking step can involve incubating the sample in normal serum from the species of secondary antibody used in the assay (e.g., for secondary antibodies raised in goat, 10% normal goat serum can be used as the blocking buffer. In Step 3, the tissue sample is incubated in the presence of a primary antibody (148) capable of recognizing a target marker on the tissue surface. In Step 4, the tissue sample bound to the primary antibody is incubated in the presence of a poly-HRP secondary antibody conjugate (150). Enzyme mediated signal amplification can be used for covalently depositing multiple fluorophores near (e.g., in the vicinity of) a targeted antigen (e.g., in a localized area on the sample surrounding the target). For example, a peroxidase, such as horse radish peroxidase (HRP), can be used to enzymatically convert a substrate for the enzyme (e.g., fluorophore or chromogen tyramides) into an activated species (e.g., tyramide or tyramide-like radicals) that can covalently bind available tyrosine residues on the sample surrounding a protein epitope targeted by the primary antibody. In Step 5, the tissue sample is incubated in the presence of hydrogen peroxide (not shown) and a set of inactive tyramides under an appropriate set of conditions (e.g., temperature, reaction time, buffer, and hydrogen peroxide concentration). Each tyramide in the set of tyramides has distinct excitation and emission properties to minimize overlapping signalsduring excitation and detection. For example, upon excitation at an appropriate wavelength of light, each set of tyramides can exhibit a unique emission maximum from about 350 nm to about 850 nm. Upon activation of the polyHRP, the inactive tyramides become activated and bind covalently to available tyrosine residues on proteins in the tissue. Non-covalently bound components remaining from Steps 1-5 then can be removed, for example, using a wash step. For example, primary and secondary antibodies can be removed from the tissue after signal development has completed without decreasing the intensity of the fluorophore deposited. After labeling has been completed, the tissue sample covalently bound to the tyramides is counterstained, for example with a nucleic acid binding dye, e.g., an indole-based dye such as DAPI (4',6-diamidino-2-phenylindole). The tissue sample then is mounted into the spatial imaging system and imaged (Step 6).

[0078] For staining of a tissue sample with two or more sets of tyramides, the tissue sample can undergo Steps 1-5 for one or more additional cycles (e g., 2-20 cycles) to produce a tissue sample that is covalently labeled with multiple (e.g., 2-20) distinct fluorophores. By way of illustration, the sample produced in Step 5 can undergo a second cycle beginning with Step 1 to identify a second marker using an appropriate primary antibody. The primary antibody used in the second cycle can be from the same or different host species (e.g., raised in rabbit or mouse) without risk of cross-reactivity. To stain the tissue sample with a second set of tyramides, tissue from Step 5 stained with a first set of detectable tyramides is subjected to a second antigen retrieval step (Step 1) to expose a second target marker on the tissue sample. The HRP -mediated reaction for each cycle can be conducted under the same set of conditions (e.g., temperature, reaction time, buffer, and hydrogen peroxide concentration) or a different set of conditions. The concentration for each set of tyramides can be adjusted to accommodate for differences between the detectable labels used for each set. Completion of a second round of Steps 1-5 results in a tissue sample that is covalently bound to two detectable labels, each distinct for a different target marker. Upon completion of Steps 1-5 of the second cycle, the labeled sample can be imaged (Step 6) to identify two distinct markers on the tissue sample.

[0079] Covalently labeling of multiple targets (e.g., greater than 6) on a tissue sample using multiple (e.g., greater than 6) iterations of a single workflow represents a significant advancement over other types of enzymatic labeling methods and kits known in the art. A particular advantage of the detectable labels described herein is that they remain covalentlybound to the tissue surface during the optional washing step of Step 5 and subsequent antigen retrieval steps during multiple iterations of the labeling process. Thus, using a single workflow including multiple cycles of enzymatic amplification, a tissue sample can be stained with multiple covalently -bound labels. Further, repeated cycles of stripping and re-probing can be conducted without significantly diminishing fluorescence signals, making the instant workflows particularly suitable for higher order multiplexing on spectral imaging systems. Spatial fhiorophores disclosed herein can provide exceptional detection of low-abundance targets, using enzyme-mediated signal amplification, and require significantly less primary antibody than other tissue labeling approaches. Covalent attachment of the fluorophores allows for primary antibody stripping in between rounds of signal amplification and permits the use of primary antibodies from the same host species, expanding the antibody options for multiplex labeling experiments. For example, primary antibodies from the same species can be used with these reagents with the cyclic stripping of antibodies between rounds of signal amplification or with an automated slide Stainer, enabling streamlined multiplex staining workflows. An advantage of the iterative labeling workflow disclosed herein is that it allows for multiplex detection of primary antibodies from the same or different host species on a tissue sample without risking antibody crossreactivity. The ability to perform mIHC using multiple primary antibodies from the same host species opens new possibilities for multiplex labeling.

[0080] Thus, provided herein are various methods for labeling a biological sample covalently with one or more populations of detectable labels. In an exemplary method, the biological sample is covalently bound to one or more populations of tyramides and then non-covalently bound components are removed from the biological sample. Each population includes a plurality of tyramides, and each tyramide in the plurality of tyramides is linked to a detectable label, as disclosed herein. The detectable labels within each population are the same. For methods using multiple populations of tyramides, the detectable labels in different populations can be unique.

[0081] In another exemplary method, a biological sample is labeled with multiple unique populations of detectable labels using iterative labeling cycles to identify multiple unique markers in the biological sample is described. A labeling cycle is represented as[I]N, wherein I is a labeling cycle and N is the number of labeling cycles, wherein N = 2-20. A labeling cycle (I) can include covalently binding a plurality of populations of tyramides to the biological sample, in an HRP -mediated signal amplification reaction, such that each population of tyramides bindsin a localized area to the biological sample in the vicinity of a marker, to provide a biological sample labeled covalently to multiple populations of detectable labels. The number of unique populations of detectable labels corresponds to the number of labeling cycles (N). The method further includes removing non-covalently bound components (e.g., components remaining from the HRP-mediated signal amplification reaction) from the biological sample to provide a biological sample covalently labeled with multiple unique populations of detectable labels.

[0082] Enzyme activated spatial amplification reagents and methods and fluorescent imaging techniques are provided that facilitate simultaneous detection of multiple biomarkers within a single tissue section and allow the deep examination of specific cellular interactions and greater characterization of cellular phenotypes. For example, enzyme-mediated signal amplification methods can be implemented for visualization of multiple (e g., 2-20) targets in one tissue sample. When bright fluorophores are used in the multiplex labeling strategy, enhanced fluorescence signal intensity can further facilitate the visualization of multiple low abundance targets in one tissue sample. Multiplex imaging assays disclosed herein using multiple labels (e.g., 4 or more) can advantageously be completed more quickly than standard techniques, e.g., in about 18 hours of less using an automated Stainer (e.g., Leica Bond Rx).

[0083] The improved techniques disclosed herein are invaluable for spatial biology research, allowing for the detection and understanding of spatial relationships and cellular interactions. In one method, quantitative image analysis is used to measure the spatial distribution of distinct cell populations based on the identified biomarkers within the tumor microenvironment. Analysis software can be used for the analysis of single cells and can provide insight to specific localization of myeloid (CD68+ cells) and lymphoid (CD3+, CD4+, CD8+ and CD20+ cells) subpopulations along with proliferating cells and extracellular matrix neighborhoods within a single tissue slice.

[0084] Many types of samples can be used in the methods disclosed herein. Samples can include a solid, for example, containing targets in a tissue slice from an organ. Samples can be derived from living matter taken from any living organism, such as an animal, such as mammals (e.g., humans), plants, fungi, archaea, or bacteria. Thus, samples can include eukaryotic cells, archaeal cells, or prokaryotic cells. The tissue sample can be from a subject selected from humans, non-human primates, rats, mice, guinea pigs, rabbits, pigs, cows, sheep, goats, horses,dogs, cats, fish, birds, reptiles, amphibians, insects, plants, fungi, bacteria, or combinations thereof.

[0085] Samples can include a cell sample, such as a cell smear or colony, or a tissue specimen derived from a living organism, such as a tissue sample from an organ. Samples can also include other naturally obtained samples such as plant tissue samples, and synthetically derived samples such as chemical or industrial products and food products.

[0086] Tissue or cell samples can be prepared by a variety of methods known to those of ordinary skill in the art, depending on the type of sample and the assay format. For instance, tissue or cell samples may be fresh or preserved, and may be, for example, flash-frozen, smeared, dried, embedded, or fixed on slides or other supports. Samples may be prepared and stained using a free-floating technique. For example, a tissue section can be brought into contact with different reagents and wash buffers in suspension or freely floating in appropriate containers, for example microcentrifuge tubes, before being mounted on slides for further treatment and examination by the methods described herein.

[0087] A tissue section can be mounted on a slide or other substrate after an incubation with immuno-specific reagents. The remains of the staining process may then be conducted after mounting. For example, for microscopic inspection in IHC and ISH, samples can include a tissue section mounted on a suitable solid substrate. For photomicrographs, sections including samples can be mounted on a glass slide or other planar substrate, to highlight by selective staining certain morphological indicators of disease states or detection of detectable targets. The substrate can be a glass or plastic microscope slide (e.g., 26 * 75 x 1 mm or 1 x 3 x 0.04 in).

[0088] For IHC, a sample can be taken from an individual, fixed, and exposed to, for example, antibodies which specifically bind to the detectable target of interest. Sample processing steps may include, for example, antigen retrieval, exposure to a primary antibody, washing, exposure to a secondary antibody (optionally coupled to a suitable detectable label), washing, and exposure to a tertiary antibody linked to a detectable label. The method also can include an autofluorescence treatment step. Washing steps may be performed with any suitable buffer or solvent, e.g., phosphate-buffered saline (PBS), Tris-buffered saline (TBS), or distilled water. The wash buffer may optionally contain a detergent, e.g., polyoxyethylene sorbitan monolaurate (TWEEN®20) or octylphenoxypolyethoxy ethanol (Nonidet P-40).

[0089] IHC samples can include, for instance: preparations including un-fixed fresh tissues and / or cells or solution samples; fixed and embedded tissue specimens, such as archived material; and frozen tissues or cells. An IHC staining procedure can include steps such as: cutting and trimming tissue, fixation, dehydration, paraffin infiltration, cutting in thin sections, mounting onto glass slides, baking, deparaffination, rehydration, antigen retrieval, blocking steps, applying primary antibody, washing, applying secondary antibody-enzyme conjugate, washing, applying a tertiary antibody conjugated to a polymer and linked with an enzyme, applying a substrate (e.g., chromogen or fluorophore substrate), washing, counter staining, applying a cover slip, and microscopic examination using a fluorescence microscope and processed using imaging and unmixing software.

[0090] ISH samples, for instance, can be taken from an individual and fixed before being exposed to a nucleic acid or nucleic acid analog probe on a recognition unit. The nucleic acid in the sample may first be denatured to expose the target binding sites. Various counterstains or paints may further be used to locate nucleic acid molecules or chromosomes within an ISH sample.

[0091] Tissue or cell samples can be fixed or embedded. Fixatives may be needed, for example, to preserve cells and tissues in a reproducible and life-like manner. Fixatives can also stabilize cells and tissues, thereby protecting them from the rigors of processing and staining techniques. For example, samples including tissue blocks, sections, or smears may be immersed in a fixative fluid, or in the case of smears, dried.

[0092] Many methods of fixing and embedding tissue specimens are known, for example, alcohol fixation and formalin-fixation and subsequent paraffin embedding (FFPE). Any suitable fixing agent can be used. Examples include ethanol, acetic acid, picric acid, 2-propanol, 3,3'- diaminobenzidine tetrahydrochloride dihydrate, acetoin (mixture of monomer) and dimer, acrolein, crotonaldehyde (cis + trans), formaldehyde, glutaraldehyde, glyoxal, potassium dichromate, potassium permanganate, osmium tetroxide, paraformaldehyde, mercuric chloride, tolylene-2,4-diisocyanate, trichloroacetic acid, and tungstic acid. Other examples include formalin (aqueous formaldehyde), neutral buffered formalin, glutaraldehyde, carbodiimide, imidates, benzoquinone, osmic acid, and osmium tetraoxide. Fresh biopsy specimens, cytological preparations (including touch preparations and blood smears), frozen sections, andtissues for IHC analysis may be fixed in organic solvents, including ethanol, acetic acid, methanol and / or acetone.

[0093] It can be useful to pre-treat the samples to increase the reactivity or accessibility of a detectable target and to reduce nonspecific interactions. If the target is an antigen, for example, a process called “antigen retrieval” may be used (and which is also known in the art as target retrieval, epitope retrieval, target unmasking, or antigen unmasking). See, Shi et al., J. Histochem. Cytochem. 45(3): 327-343 (1997). Antigen retrieval encompasses a variety of methods including enzymatic digestion with proteolytic enzymes, such as proteinase, pronase, pepsin, papain, trypsin, or neuraminidase. Heat can be used, such as heat-induced epitope retrieval or HIER. Heating can involve microwave irradiation, a water bath, a steamer, a regular oven, an autoclave, or a pressure cooker in an appropriately pH stabilized buffer, usually containing EDTA, EGTA, Tris-HCl, citrate, urea, glycin-HCl, or boric acid. Detergents can be added to the HIER buffer to increase the epitope retrieval, or to the dilution media and / or rinsing buffers to lower non-specific binding. Combinations of different antigen retrieval methods may be used. The antigen retrieval buffer may be aqueous, but may also contain other solvents, including solvents with a boiling point above that of water such as glycerol. This allows for treatment of the tissue at greater than 100 °C at standard pressure.

[0094] Signal-to-noise ratio can be increased by different physical methods, including application of vacuum, ultrasound, or freezing and thawing tissue samples before or during incubation of the reagents.

[0095] Treatments can be performed to reduce nonspecific binding. For example, carrier proteins, carrier nucleic acid molecules, salts, or detergents may reduce or prevent non-specific binding. Non-specific binding sites may be blocked in some embodiments with inert proteins like, HSA, BSA, ovalbumin, with fetal calf serum or other sera, or with detergents like polyoxyethylene sorbitan monolaurate (TWEEN®20), octylphenoxypolyethoxyethanol (Nonidet P-40), / -octylphenoxypolyethoxyethanol (TRITON™ X-100), triterpene glycosides (Saponin), nonionic polyoxyethylene surfactants (BRIJ®-35), or nonionic triblock copolymers (PLURONICS®). Alternatively, non-specific binding sites can be blocked with unlabeled competitors for the recognition event between the target and the affinity molecule. For example, in the case of a nucleic acid interaction, non-specific binding may be reduced by adding unlabeled competitor nucleic acids or nucleic acid analogs such as digested, total human DNA,or unlabeled versions of the affinity molecule. In addition, repetitive sequences may be blocked, for example, using nucleic acids or nucleic acid analogs that specifically recognize those sequences, or sequences derived from a total DNA preparation. Salt, buffer, and temperature conditions may also be modified to reduce non-specific binding.

[0096] Cross reactivity of different components of the detection methods can be avoided, for example, by using antibodies derived from different species. Furthermore, combinations of, for example, secondary antibodies against primary antibodies and haptens can also be used to avoid unwanted cross reactivity. Endogenous biotin binding sites or endogenous enzyme activity (for example phosphatase, catalase, or peroxidase) can be removed as a step in the staining procedure. Endogenous biotin and peroxidase activity may be removed by treatment with peroxides, while endogenous phosphatase activity may be removed by treatment with levamisole. Heating may destroy endogenous phosphatase and esterase activity.

[0097] Biological samples such as cells and tissues can be labeled with a wide variety of detectable labels, as disclosed herein. For spectral imaging applications, biological samples can be stained with fluorophores. Suitable detectable labels for use in spatial imaging applications include fluorophores capable of emitting light in the UV (e.g., UVA) to IR spectral range upon excitation at an appropriate wavelength of light (e.g., about 350 nm to about 850 nm). Spatial imaging applications can utilize fluorophores with emission profiles in the visible to near-IR spectral region (e.g., about 400 nm to about 850 nm). In certain embodiments, the detectable label is an infrared- emitting (IR) dye. Fluorescent dyes that function in the near infrared (NIR) and IR region can have facilitate imaging with minimal autofluorescence from biological samples, reduced light scattering, and high tissue penetration. In certain embodiments, the detectable label is a fluorophore that exhibits light with an emission maximum in the far-red or near-IR spectral region upon excitation at an appropriate wavelength of light.

[0098] Detectable labels useful for staining cells and tissues can be in the form of a conjugate. For example, a detectable label, such as a fluorophore, chromogen, or hapten can be covalently bound to a molecule or group that can bind (either covalently or non-covalently) to a target. For example, a fluorophore can be covalently bound to a protein (e.g., antibody) to provide a fluorescently-labeled protein conjugate. In certain methods, a fluorophore can be conjugated to an affinity molecule, such as an antibody or biotin, that can bind to a target molecule non-covalently (e.g., antigen or streptavidin). Alternatively, fluorophores can beconjugated (either directly or through a linker) to a substance that can covalently bind or associate with the biological sample. For example, a fluorophore bearing an azide or alkyne functional group can bind covalently via a cycloaddition reaction (e.g., click reaction) to a biological substance that bears a complementary alkyne or azide reaction partner (e.g., protein).

[0099] In another example, a detectable label can be covalently bound to a tyramine group to provide a tyramide conjugate (also referred to interchangeably herein as a “tyramide”). A tyramide conjugate is capable of binding covalently under the appropriate conditions to an available tyrosine group on or in the biological sample, thereby covalently linking the detectable label (e.g., fluorophore, chromogen, hapten, and the like) to the sample. In certain embodiments, a fluorophore can be covalently bonded (e.g., directly or indirectly linked) to a tyramine group to provide a fluorescently-labeled tyramide conjugate.

[0100] Detectable labels for biological applications, which are frequently performed in aqueous environments, can further include groups for improved water-solubility. For example, derivatives of detectable labels, such as a fluorophore or chromogen, can be substituted with one or more poly(ethylene glycol) (PEG) and / or sulfonate groups to increase the water-solubility of the detectable label.

[0101] Representative examples of suitable fluorophores for imaging application include, without limitation fluorescein or its derivatives, such as fluorescein-5-isothiocyanate (FITC), 5- (and 6)-carboxyfluorescein, 5- or 6-earboxyfluorescein, 6-(fluorescein)-5-(and 6)-carboxamido hexanoic acid, fluorescein isothiocyanate, xanthene and its derivatives; rhodamine and its derivatives, such as tetramethylrhodamine and tetramethylrhodamine-5-(and-6)-isothiocyanate (TRITC); cyanine and its derivatives,; coumarin and its derivatives, such as (diethyl- amino)coumarin or 7-amino-4-methylcoumarin-3 -acetic acid, succinimidyl ester (AMCA), as well as BODIPY dyes, pyrene-based dyes, anthracene-based dyes , as well as derivatives thereof, and non-standard dye scaffolds such as charge transfer dyes that exhibit long-Stokes shift behaviors.

[0102] Fluorophores for use in imaging application can include reactive functional groups (e.g., amine, carboxylic acid, azide, alkyne, succinimidyl ester, sulfonyl chloride, maleimide, and the like) that are capable of reaction with reactive group in or on a substance to provide a fluorescently-labeled substance. Representative examples of reactive fluorophores include, but are not limited to: sulforhodamine 101 sulfonyl chloride (TexasRed™ or TexasRed™ sulfonylchloride; 5-(and-6)-carboxyrhodamine 101, succinimidyl ester, also known as 5-(and-6)- carboxy-X-rhodamine, succinimidyl ester (CXR); lissamine or lissamine derivatives such as lissamine rhodamine B sulfonyl chloride (LisR); 5-(and-6)-carboxyfluorescein, succinimidyl ester (CFI); fluorescein-5-isothiocyanate (FITC); 7-diethylaminocoumarin-3 -carboxylic acid, succinimidyl ester (DECCA); 5-(and-6)-carboxytetramethylrhodamine, succinimidyl ester (CTMR); 7-hydroxycoumarin-3-carboxylic acid, succinimidyl ester (HCCA); 6-fluorescein-5- (and-6)-carboxamidolhexanoic acid (FCHA); A-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza- 3 -indacen epropionic acid, succinimidyl ester; also known as 5,7-dimethyl BODIPY™ propionic acid, succinimidyl ester (DMBP); “activated fluorescein derivative” (FAP), available from Thermo Fisher Scientific; eosin-5-isothiocyanate (EITC); erythrosin-5-isothiocyanate (ErlTC); and Cascade™ Blue acetylazide (CBAA) (the O-acetylazide derivative of 1 -hydroxy-3, 6, 8- pyrenetri sulfonic acid). Yet other potential fluorophores useful herein include, but are not limited to, fluorescent proteins such as green fluorescent protein (GFP) and its analogues or derivatives, fluorescent amino acids such as tyrosine and tryptophan and their analogues, and fluorescent nucleosides.

[0103] Fluorescent molecules for use as detectable labels include commercially available compounds or their reactive counterparts, such as, for example, cyanine dyes such as Cy2, Cy3, Cy 3.5, Cy5, Cy5.5, Cy 7 from Cytiva (Marlborough, MA) ; cyanine and rhodamine-based DY dyes from Dyomics GmbH (Germany); and dyes that are commercially available from Sigma- Aldrich Co. (St. Louis, MO) and Thermo Fisher Scientific (Waltham, MA), including, e.g., eFluor™ dyes, Alexa Fluor™ dyes (e.g., Alexa Fluor™ 350, 488, 555, 568, 594, 647, 680 and 750), Alexa Fluor™ Plus dyes, NovaFluor™ dyes, Oregon Green 488, Pacific Blue (3-carboxy- 6,8-difluoro-7-hydroxycoumarin), and Rhodamine Green. Additional commercial suppliers of fluorophores and reactive versions thereof suitable for spatial imaging techniques, as disclosed herein, include the iFluor and mFluor reagents, as well as PE-Cy5 and APC-Cy7 tandem fluorescent probes from AAT Bioquest and ATTO fluorescent labels from Atto-Tec GmbH (Siegen, Germany).

[0104] Yet other examples of detectable labels that can be used in imaging methods described herein include phycoerythrin and inorganic fluorescent labels such as particles based on semiconductor material (e.g., Qdot™ semiconductor nanocrystals from Thermo Fisher Scientific).

[0105] Methods disclosed herein can implement fluorescently-labeled tyramide and tyramide-like reagents. Examples of commercially available fluorescently-labeled tyramide and tyramide-like reagents include CF™ dye-labeled tyramides from Biotium (Fremont,CA); iFluor® Styramide™ reagents (e.g. iFluor 350, 488, 546, 555, 568, 594, 647, 60, 700, and 750 Styramide™) from AAT Bioqust (Fremont, CA); Opal™ 520, 540, 570, 620, 650, and 690 reactive fluorophores from Akoya Biosciences; Alexa Fluor™ tyramides and Aluora™ Spatial Dyes (e.g., Aluora 430, 488, 514, 555, 594, 647, 700, and 750 Spatial Dyes) from Thermo Fisher Scientific (Waltham, MA).

[0106] Methods disclosed herein also can implement colometric tyramide and tyramide-like reagents. For example, for colorometric imaging, the SuperBoost™ EverRed and EverBlue Colorimetric HRP Kits from Thermo Fisher Scientific can be used with enzymatic amplification methods, such as disclosed herein.

[0107] Where commercial versions of fluorescent tyramide reagents are not available, tyramides can be synthesized from a wide variety of commercially-available, reactive versions of fluorophores reagents described herein. For example, fluorescent tyramides that can be excited and / or emit across the UV-visible to near-IR spectral region can be prepared with fluorophores including, without limitation, cyanine and rhodamine-based dyes or water-soluble derivatives thereof, such as those available from Cytiva (e.g., Cy 2, Cy3, Cy 3.5, Cy5, Cy5.5, Cy 7);Dyomics GmbH (e.g., DY-675, DY-676, DY-677, DY-678, DY-679P1, DY-680, DY-681, DY- 682, DY-684, DY-700 - DY706, DY-720, DY-730 - DY-734, DY-736, Dy-747P1, DY-749 - DY- 752, and the like); and AAT Bioquest (e.g., iFluor® reactive dyes, such as iFluor 350, 405, 430, 450, 488, 514, 532, 546, 555, 594, 560, 647, and 700).

[0108] Described herein are workflows for use with immunology targets to characterize how cells interact across complex tissues imaged on a high-resolution spatial slide scanner.Workflows provided herein utilize one or more labeling strategies to facilitate antibody-based detection of these targets. The study of molecules in a two-dimensional or three-dimension context involves a variety of spatial biology techniques to visualize molecules within individual cells and tissues. For example, the field of spatial omics can offer users high-throughput solutions and insights around spatial organization and cell types. Spatial biology techniques include, for example, the use of antibody -based imaging spatialomics studies. Antibody based spatialomics studies often require the detection of a panel of markers consisting of high, medium and lowabundant protein markers. The detection of low abundant markers requires signal amplification which is typically performed one at a time and iteratively. As a result, the workflow for detection of multiple antibody targets on a single FFPE tissue is time consuming. The disclosed workflows address issues associated with existing workflows and can enable higher throughput of tissues.

[0109] Iterative labeling of protein markers with primary antibodies, followed by secondary antibody-enzyme conjugation, and then covalent deposition of an enzymatic fluorescent substrate, can be used to evaluate low, medium, and high abundance protein markers. Once a stable signal is deposited, antibodies can be stripped off and the tissue is re-probed for the second abundant protein target with a new primary antibody / secondary antibody-enzyme conjugate having a different fluorescent signal deposition. This protocol is repeated until all abundant markers are detected with distinct fluorescent signal deposition. Next, protein markers are detected with a cocktail mixture including antibodies each directly labeled with a distinct fluorescent dye. These are added at one time to the tissue and incubated as the last step prior to or with a nuclear stain. Individual incubation steps can be eliminated using instrumentation that can provide iterative incubation of reagents needed for abundant protein detection followed by the one-time incubation of the cocktail of directly labeled markers.

[0110] Methods provided herein can reduce the time required for the detection of a panel of antibody markers on a tissue section. The methods provide for the combination of steps that are mutually compatible for the detection of both high, medium, and low abundance markers on a single tissue section. The instant methods address issues with existing workflows commonly used in the spatial omics field. Spatial omics studies can make use of panels designed for high levels of multiplexing of fluorescently based signals that are spectrally deconvoluted to define the localization and density of individual protein targets. This aggregate information of multiple target distribution provides useful information on the biology of the sample including the context of the local (microenvironment) of the tissue. Individual detection of a single protein marker does not define the complete biology. However, combining signals from multiple protein markers is difficult and time consuming. The individual signals need to be adjusted for optimal detection, and high and low abundant protein markers and need different methods to create signals that can be detected together. This problem is addressed by streamlining the labeling process so that low abundant markers can be successively amplified without crosstalk using compatible reagents in the method. The high and moderate signals can be deposited in a singletreatment using directly labeled dye-antibody conjugates without the time-consuming limitations of iterative labeling with at most two species of antibodies (mouse and rabbit) without the need to strip and re-probe as would be required for each of the antibodies of the panel.

[0111] The spatial imaging workflow can include an enzyme-mediated signal amplification technique, which is a highly sensitive method for detection of low-abundance targets in fluorescent immunocytochemistry (ICC), immunohistochemistry (IHC), and in situ hybridization (FISH) applications. Standard enzyme-mediated amplification methods use horseradish peroxidase (HRP)-catalyzed deposition of a labeled tyramide substrate in situ on and near a target protein or nucleic acid sequence, where tyramides can be labeled with a fluorophore, chromogen, or a hapten such as biotin, DNP, or DIG.

[0112] FIG. 2 illustrates a single cycle of a representative labeling workflow including enzyme-mediated signal amplification. In a cycle of the workflow, a marker in the sample is labeled with a primary antibody that recognizes a marker in the sample. The sample then is treated with a secondary antibody conjugate that can recognize and bind to the primary antibody. The secondary antibody is conjugated to one or more peroxidase groups (e.g., HRP). As used herein, HRP is used interchangeably herein to refer to a single HRP molecule or polyHRP. Poly- HRP -mediated tyramide labeling reactions can be particularly advantageous for detection of low abundant targets in multiplexable fluorescent immunocytochemistry (ICC), immunohistochemistry (IHC), and in situ hybridization protocols. A labeling reagent is added (e.g., a tyramide substrate) and then activated in the presence of the peroxidase. In the presence of low concentrations of H2O2, HRP can convert the tyramide substrate into a reactive form that can covalently bind to tyrosine residues on proteins in the vicinity of the enzyme (i.e., on and surrounding the protein epitope targeted primary antibody). The sample then is subjected to a stripping step to remove components that are not covalently bound to the sample (e.g., antibodies and non-reacted labeling reagents). The stripping step does not remove labeling reagent that is covalently bound to the sample. Once the tissue is labeled with a first set of labeling reagents, the workflow can be repeated. Multiple, sequential staining iterations can be used to label the tissue with two or more types of labeling reagents.

[0113] The iterative HRP -mediated signal amplification labeling method described includes various advantages over standard antibody labeling methods. The high sensitivity of the enzymatic labeling system is useful for detection of low abundance targets as a result of highdensity tyramide labeling of the tissue. HRP-mediated signal amplification also can increase processing speed relative to manual approaches, especially when labeling samples with multiple antibodies. For example, HRP-mediated labeling in a multiplex (e.g., 8-plex) assay, as disclosed herein, on an automated Stainer can take ~12 hours; whereas manual staining can take about ~2-3 days. Iterative labeling with fluorescent signal deposition is resistant to the stripping even after multiple antigen retrieval steps, thereby reducing protocol complexity, without cross-target or off target signal development.

[0114] Thus, a general method is provided herein in which a biological sample is covalently bound to a detectable label. In certain embodiments, the detectable label includes a fluorophore, chromogen, or hapten. The detectable label can be bound to the sample using the enzyme- mediated amplification process. Upon enzymatic activation under the appropriate set of conditions, the tyramide binds to the biological sample in a localized area near (i.e., in the vicinity of) the marker to provide a biological sample labeled covalently to the first detectable label. The biological sample then can be treated to remove non-covalently bound components remaining after the amplification step.

[0115] While standard HRP-mediated amplification methods typically employ tyramide reagents to detect low-abundance targets in cells and tissues, conjugates of certain tyramide replacement compounds have demonstrated several advantages over traditional tyramide reagents when implemented in these types of amplification assays. When used in enzyme- mediated amplification workflows, as described herein, styramide conjugates can use less primary antibody than tyramide-based amplification, while maintaining the same level of sensitivity. Styramide conjugates are also capable of producing a fluorescence signal that is significantly higher than standard tyramide reagents. Because styramide radicals are more reactive than tyramide radicals, labeling with styramides also can be faster and more robust. Further, styramide-based reagents can achieve improved resolution as styramide radicals, deposited close to the HRP-target site during the enzyme-mediate workflow, can exhibit minimal loss of resolution due to diffusion.

[0116] For spatial biology imaging applications, polyHRP -mediated tyramide-based labeling strategies have been used for staining biological samples using a limited number of detectable labels. Expansion of high order multiplex labeling, however, has been limited due to theavailability of appropriate compounds and methods, especially for detection outside of the visible range of the electromagnetic spectrum (e.g., far-red to near-IR).

[0117] Various commercially-available kits are available for labeling tissue samples with fluorophores using enzyme-mediated signal amplification methods, including the Tyramide SuperBoost™ Kits from Thermo Fisher Scientific, Tyramide Amplification Kits from Biotium, (Fremont, CA), and the Opal™ 6-Plex Detection Kits from Akoya Biosciences (Marlborough, MA). To date, however, commercial kits that implement fluorescent tyramides in an enzyme- mediated amplification workflow under the same conditions can achieve fluorescent labeling of up to 6 targets on a tissue (not including DAPI counterstain).

[0118] The Opal TSA assay kit, for example, achieves multiplex labeling using a series of fluorescent dyes having emission maxima ranging from 480 nm to 690 nm. Labeling of an 8thtarget using a reagent with emission maximum of 770 nm is achieved through a two-step antibody-mediated reaction that deposits a fluorophore (i.e., Opal Polaris 780) non-covalently on the tissue. In this process, a tissue is treated with DIG-tyramide, amplified in the presence of HRP, and then subsequently treated with fluorescently-labeled, primary anti-DIG (digoxigenin) antibody. The manufacturer cautions the user that the Opal Polaris 780 reagent must go last and that no antigen removal steps should be performed after incubation of the reagent with the tissue. The requirement of a separate multi-step process to label a target with the Opal Polaris 780 dye adds significant cost, complexity, and increases throughput time.

[0119] In contrast to previous methods requiring a cumbersome two-step labeling involving binding and enzymatic amplification of a DIG-tyramide with subsequent anti-DIG primary antibody binding for deposition of fluorophores, methods are provided herein that implement multiple (e.g., 8 or more) fluorescent tyramide conjugates of fluorescent dyes for identifying multiple targets (e.g., 8 or more) on a tissue using an enzyme-mediated amplification method. In this method, each of the fluorescently-labeled tyramides are covalently bound to the available tyrosine groups in the vicinity of unique markers on the tissue.

[0120] Described in the present disclosure are labeling strategies that can be used to significantly expand the number of targets that can be imaged using modern spatial biology imaging techniques. For example, labeled tyramides are described herein that can be used to effectively label biological samples (e.g., tissues or cells) with multiple types of bright detectable labels to permit high-fidelity multiplexing of a variety of validated antibody clones.

[0121] Thus, provided herein are methods that include multiple cycles (i.e., rounds) of staining a biological sample with detectable labels, where each cycle includes removal of non- covalently bound components ahead of a subsequent labeling step. Non-covalently bound components can include components from labeling, washing, and antigen retrieval steps (e.g., antibodies, enzyme-antibody conjugates, tyramides, and the like). A single labeling round can include antigen retrieval to unmask a marker of interest in the sample, covalent binding of a set of detectable labels (e.g., tyramides) in the vicinity of the marker (e.g., using an HRP -mediated signal amplification reaction), and removal of non-covalently bound components from the biological sample remaining from the antigen retrieval and / or amplification reaction.

[0122] In certain embodiments, the detectable label includes a fluorophore that emits light in the UV / Vis to near-IR spectral region upon excitation at an appropriate wavelength of light. For effective multiplex labeling, the fluorescence emission maximum of each of the fluorophores should be different and spectrally resolved. The steps of the method can be repeated in a sequential manner to provide a biological sample labeled covalently with multiple sets of detectable labels, where each set of detectable labels is localized in the area surrounding the marker of interest. For example, the method can be repeated 2-20 times to provide a biological sample labeled covalently with 2-20 unique detectable labels to identify 2-20 different markers of interest, respectively.

[0123] Spectral images of particularly high quality can be achieved with spectral unmixing when all fluorophores used in a multiplex experiment exhibit similar fluorescence intensities. A spatial imaging panel including multiple (e.g., 5 or more) fluorophores, where all fluorophores in the panel exhibit similar fluorescence intensities can be prepared by evaluating pairs of fluorophores emitting in different channels of the instrument. By way of example, a pair of fluorophores with emission profdes that are detectable in adjacent channels of the imaging instrument can be evaluated to determine whether the fluorescence intensity of each fluorophore is similar. The evaluation of a pair and panel of fluorophores should be conducted under the same staining and imaging data processing conditions to control variability. Two fluorophores are considered to have similar fluorescence intensity if the fluorescence intensity of one dye is no greater than 5 times the fluorescence intensity of the second dye (i.e., the fluorescence intensities of the first and second dyes differ by a factor of 5 or less), under the same imaging and processing conditions. Depending on the pairs of fluorophores, the fluorophores can exhibitfluorescence intensities that differ by a factor of less than 5 times (e.g., less than 4 times; or less than 3 times; less than 2 times; or less than 1 time). Engineering the panel to include dyes that have similar fluorescence intensities and exhibit discrete emission profiles can minimize the extent of bleed-through between channels, resulting in production of extremely high-quality spectral images. In certain embodiments, where sets of detectable labels include sets of different fluorophores, the concentrations of each set of fluorophores can be adjusted to control the relative fluorescence intensities between the sets of labels. For example, the concentrations of each set of fluorophores used in a labeling strategy can be adjusted such that upon detection the fluorescence signal intensities for two sets of fluorophores differ by a factor of 5 or less.

[0124] The instant methods provide a distinct advantage over prior methods because all fluorophores in the panel are bound covalently to the tissue surface, such that the bound fluorescent labels are not readily removed in subsequent washing or heating steps commonly used in antigen retrieval processes. Consequently, the detectable labels can be bound to the tissue without regard for the order of deposition. When used in advanced spectral imaging systems, sample stained with multiple unique fluorophores according to methods disclosed herein can generate high intensity emission signal at wavelengths that span across the UV / Vis to near-IR spectral region and are spectrally resolved. Detection of greater than 6 unique markers, including detection of at least one type of fluorophore that emits in the far-red or near-IR spectral region (e.g., greater than 710 nm) using a multiple cycles of an enzyme-mediated amplification workflow represents a significant advancement in the field of spatial imaging.

[0125] The quality of images produced using advanced spectral imaging equipment can be enhanced by appropriate selection of the detectable labels used for the multiplex panel. For example, fluorophores can be selected based on the spectral profiles of each fluorophore, as well as their relative fluorescence intensities. For example, a multiplex panel can be assembled by including multiple unique fluorophores that each exhibit a unique fluorescence maximum ranging from about 350 nm to about 850 nm upon appropriate irradiation by the spectral imaging system. For use in spectral imaging systems equipped with spectral unmixing capabilities, it can be advantageous to construct a multiplex panel using fluorophores that exhibit similar relative fluorescence intensities to ensure that minimal fraction bleed through of other dyes remains after applying linear unmixing algorithms. For example, fraction bleed through can be minimizedwhen fluorophores detectable in neighboring channels of a spectral imaging system exhibit similar fluorescence signal intensities.

[0126] In certain embodiments, a set of fluorophores, as shown in Table 1, with each fluorophore in the form of a conjugate with a tyramide or tyramide-like group that can bind covalently to the surface of a biological sample (e.g., tissue or cell) through an HRP-mediated process, as disclosed herein, in a single step. The same set of conditions and reagents can be used to activate and covalently bind each of the fluorophores in the set to the sample. Enzyme inactivation and antibody stripping can be performed immediately after labeling with the fluorophores to ready the sample for subsequent signal amplification reactions. Further, there is no requirement that the sample be labeled with the fluorophores in a particular order. A multiplex fluorophore set, such as described with reference to Table 1, for covalently labeling a single tissue or cell sample and that includes fluorophores that can be excited and detected in the far-red and near-IR channels of the spectral imaging system and can withstand the rigors of multiple stripping and re-probing steps offers a significant improvement over existing multiplex kits currently on the market.

[0127] Further methods are disclosed herein for labeling multiple markers on a tissue sample using a combination of enzyme-mediated amplification to identify markers of low-medium abundance followed by treatment with a mixture of antibodies to identify markers of high abundance. Referring to FIG. 3, an exemplary workflow is depicted that employs four (4) rounds of HRP- mediated tissue labeling to investigate four low-medium abundance markers. The covalently-attached labels are not removed during the four stripping and re-probing steps (Rounds 1-4). Subsequent treatment with an antibody cocktail can be used to investigate the four (4) high abundance markers. The exemplary workflow is appropriate for assessing low, medium and high abundance markers in a single experiment.

[0128] In another aspect, a workflow is provided for identifying multiple (e.g., two or more) markers in a tissue sample. Workflows described herein can be used to identify between 2 to 20 targets in a tissue sample, although higher levels of multiplexing using available spectral imaging systems can theoretically be achieved through selection of appropriate detectable labels and data processing techniques. Workflows described herein readily can be used to achieve detection of 6 or more markers in a tissue sample. In some embodiments, workflows disclosed herein can be used for detection of 7, 8, or 9 unique markers in a tissue sample. In some embodiments,workflows disclosed herein can be used for detection of 8-20 unique markers in a tissue sample. In some embodiments, 9-15 unique markers can be detected in a tissue sample using workflows disclosed herein.

[0129] The workflows disclosed herein can be highly effective for labeling biological samples with multiple detectable labels. A biological sample can be produced using the instant methods that is labeled with two or more detectable tyramides. For example, two or more sets of detectable tyramides can be covalently bound to the sample in a localized area to the biological sample in the vicinity of different markers in the biological sample. In certain embodiments, each set of detectable tyramides includes a different fluorophore. For example, a first set of tyramides can include a fluorophore that emits light with an emission maximum between 350 nm and 710 nm and a first fluorescent signal intensity upon excitation at an appropriate wavelength of light. The biological sample also can be labeled with a second set of tyramides that include a different fluorophore that emits light with an emission maximum between 710 nm and 850 nm and a second fluorescence signal intensity. In certain embodiments, the fluorescence intensities of the two populations of fluorophores differ by a factor of 5 or less.

[0130] Also provided are biological samples that are labeled with 2 or more unique detectable sets of tyramides, wherein each set includes a spectrally unique fluorophore. In certain embodiments, the biological sample is labeled with eight or more sets of detectable tyramides, wherein each set includes a spectrally unique fluorophore. For example, a first set of detectable tyramides includes tyramides that are each linked to a first fluorophore; a second set of detectable tyramides including tyramides that are each linked to a second fluorophore, and so forth, where the different (e.g., first, second, etc.) fluorophores are spectrally unique. For use in an imaging system, the biological sample can be mounted on an imaging support, such as a microscope slide, cuvette, well or dish. The biological sample is typically mounted on the imaging support in a mounting medium. In certain embodiments, the biological tissue is embedded in a mounting medium, wherein the mounting medium has a refractive index of 1.47 - 1.52.

[0131] Also provided herein are compositions that include two or more populations of tyramides. A representative composition can include a first population of tyramides at a first concentration, wherein the first population includes a plurality of fluorophores capable of emitting light with an emission maximum between 350 nm and 710 nm and a first fluorescentsignal intensity. The composition further includes a second population of tyramides at a second concentration (either the same or different from the concentration used for the first population of tyramides), wherein the second population includes a plurality of second fluorophores capable of emitting light with an emission maximum between 710 nm and 850 nm and a second fluorescence signal intensity. The concentrations of tyramides can be adjusted to adjust the intensity of the fluorescence signal that is detected. For example, in certain compositions, the concentrations of the two tyramide populations are adjusted such that the first and second fluorescence intensities differ by a factor of 5 or less.

[0132] Also provided herein are kits for labeling multiple (e.g., two or more) markers in a biological sample (e.g., tissue or cells). An exemplary kit can include reagents and instructions for labeling multiple markers using an HRP -mediated signal amplification process. Typically, the user can supply primary antibodies specific for detected desired markers in the sample. A representative kit designed for labeling multiple markers on a biological sample (e.g., a tissue or cell) can include polyHRP-conjugated secondary antibodies from the desired species (e.g., mouse, goat, and the like), hydrogen peroxide, blocking buffer (e.g., goat serum), reaction buffer, and two or more sets of detectable (e.g., fluorescently-labeled) tyramide substrates; and instructions for labeling and detecting multiple markers in a biological sample. In certain embodiments, the kit includes eight (8) or more sets of detectable (e.g., fluorescently-labeled) tyramide substrates and appropriate polyHRP secondary antibody conjugates and reagents to achieve multiplex labeling of a biological sample.

[0133] The following non-limiting examples further describe various compounds, methods, compositions, uses, and embodiments disclosed herein.EXAMPLESMATERIALS AND METHODS

[0134] Spectral imaging was performed on an EVOS SI 000 Spatial Imaging System (Thermo Fisher Scientific) was used to capture high-resolution images. The imaging system utilizes advanced fluorescence microscopy technology that is capable of rapidly capturing images across a wide variety of channels and enables spectral unmixing for high multiplex sample analysis (<1 hour / cm2). The instrument is equipped for multiplex spectral fluorescence,transmitted brightfield, phase-contrast, and color brightfield and includes a highly intuitive graphical user interface. Other features of the system include multiplex spectral unmixing; capable of imaging up to 4 tissue slides in one sitting; includes robust and fast laser based autofocus; and can provide precise visualization of cellular structures.

[0135] Primary antibody conjugates described herein were validated across various tissue types, including, but not limited to, normal and certain cancerous human tissues: spleen, appendix, duodenum, tonsil, thymus, cerebellum, liver, colon, and the like.EXAMPLE 1: MULTIPLEX LABELING OF TISSUE WITH TYRAMIDES

[0136] FFPE human tonsil tissue was labeled to identify 8 targets according to methods disclosed herein uses a set of 9 fluorophores. The set of 9 fluorophores included DAPI (B) and 8 different fluorescent dye labels were used identify markers on the tissues. The fluorescent dyes in this panel were used at a concentration such that each dye exhibited a similar relative fluorescence intensity. Excitation / emission wavelengths (nm) and corresponding markers corresponding to panels (C) - (I): (C) 427 / 499 (PDL1); (D) 493 / 518 (CD4); (E) 512 / 529 (eCad); (F) 553 / 567 (CD8a); (G) 589 / 615 (PD1); (H) 652 / 670 (CD3d / e; (I) 687 / 706 (SMA); (J) 757 / 783 (CD20). As shown in the images of FIG. 4, the 9-plex tissue labeling scheme facilitated investigation of the immune responses within the human tonsil and differentiate between T cells, B cells, and tissue structure.

[0137] In a separate experiment, human duodenum tissue was labeled using 9 fluorophores, as described above, and primary antibody conjugates containing mouse, rabbit, and biotin secondary antibodies and imaged using the above fluorophores to identify targets in the tissue (FIG. 5). The image in (A) illustrates three different primary antibody detection strategies that can be multiplexed together on a single sample. Sample was stained covalently with fluorescent labels using HRP -mediated amplification against biotinylated primary antibody against CD8a (detected with Dye 488 seen in green), rabbit primary antibody against CD3e (detected with Dye 555 seen in red) and mouse primary antibody against CD68 (detected with Dye 647 seen in white) and counterstained with DAPI. The image shows that different primary antibodies detection strategies are compatible and therefore can be used on a single sample. The image in (B) shows that a single type of primary antibody can be multiplexed together on a single sample, in this case, mouse primaries were detected using the mouse secondary HRP. The sample wasstained with fluorescent labels using HRP -mediated amplification against mouse primary antibody against PCNA (detected with Dye 514 seen in green), mouse primary antibody against Vimentin (detected with Dye 594 seen in red) and mouse primary antibody against SMA (detected with Dye 700 seen in white) and counterstained with DAPI. The image shows that a single type of mouse primary antibody can be detected using mouse secondary HRP using multiple rounds of staining in a single sample. The image in (C) is for a sample treated with two rounds of tyramide labeling mouse and streptavidin secondary antibodies, followed by labeling with two primary antibody conjugates. Sample was stained with fluorescent labels against biotinylated primary antibody against Ki67 (detected with Dye 488 seen in green), mouse primary antibody against PCNA (detected with Dye 555 seen in red) and primary antibody conjugates against CD45RO Alexa Fluor Plus 647 (seen in white) and SMA (AE1 / AE3) Alexa Fluor Plus 750 (seen in blue). The image demonstrates that multiplex staining can be achieved on a single sample using multiple rounds of tyramide labeling, along with labeling with primary antibody conjugates.EXAMPLE 2: LABELING OF TISSUE WITH PRIMARY ANTIBODIES

[0138] A general method for labeling a sample in a single step using a primary antibody conjugate mixture, where each antibody is labeled with a different organic fluorophore is depicted in FIG. 6. The labeling method can be used to label tissue samples using various fluorophores conjugated to validated IHC antibody clones. For example, fluorophores, such as Alexa Fluor, Alexa Fluor Plus, and eFluor dyes (Thermo Fisher Scientific) can be conjugated to antibody clones for labeling of a tissue sample.

[0139] Normal human tonsil tissue samples were labeled with multiple dye labeled primary antibody conjugates according to the method described in FIG. 6. Primary antibody conjugates were confirmed and evaluated against secondary antibody IHC labeling. Images of the labeled samples are shown in FIG 7. (A) PanCK (cytokeratin), epithelial marker labeled with Alexa Fluor™ 514 on normal human tonsil tissue. Full tissue stitch imaged as main figure and zoomins correspond to one 20X field of view with primary antibody labeling (top) and primarysecondary antibody labeling (bottom) methods. (B) CD68, macrophage marker labeled with Alexa Fluor™ Plus 488 on normal human tonsil tissue. Full tissue stitch imaged as main figure and zoom-ins correspond to one 20X field of view with primary antibody labeling (top) andprimary-secondary antibody labeling (bottom) methods. (C) FoxP3, transcriptional regulator marker labeled with Alexa Fluor™ Plus 647 on normal human tonsil tissue. Full tissue stitch imaged as main figure and zoom-ins correspond to one 20X field of view with primary antibody labeling (top) and primary-secondary antibody labeling (bottom) methods.In a separate experiment, FFPE human colon adenocarcinoma was labeled using primary IHC validated antibody conjugates and imaged (see, FIG. 9). A 5-plex (DAPI included) tissue labeling method can allow investigation of the immune responses within the human colon and differentiate between T cells, tissue structure, and proliferative cells. FIG. 9 shows a composite image (A) of tissue labeled with multiple labels, where images for individual channels are shown in B-E. (B) PanCK (AE1 / AE3) eFluor™ 506 (seen in green); (C) CD8a (C8 / 144B) Alexa Fluor™ 594 (seen in red); (D) Ki67 (SolA15) Alexa Fluor™ 514 (seen in yellow), and (E) SMA (1 A4) Alexa Fluor™ 700 (seen in blue).EXAMPLE 3: LABELING WITH AN ANTIBODY PANEL

[0140] Multiple primary antibody conjugates were combined in a single mixture for multiplexing on a single normal human tonsil tissue sample. FFPE human tonsil tissue was labeled with antibody panel including high and moderate abundant markers of PanCK (AE1 / AE3), CD20, CD68 and Ki-67. A spectrally unmixed image of a normal human tonsil labeled with a mixture of primary antibody conjugates against PanCK (AE1 / AE3) Alexa Fluor™ 700 (seen in red), CD20 (L26) Alexa Fluor™ Plus 750 (seen in purple), CD68 (KPI) Alexa Fluor™ 488 (seen in blue) and Ki67 (SolA15) eFluor™ 506 (seen in green) is shown in FIG. 8.

[0141] In a separate experiment, differences in FFPE human tonsil tissue types were evaluated using IHC validated primary antibody dye conjugates and imaged using M7000 Imaging System (see, FIG. 14). In each of the images, PanCK (AE1 / AE3) is seen in green; CD20 (L26) is seen in red; and DAPI is seen in blue. The images can be used to visualize and quantify cellular traits of innate and adaptive immune cell populations that can be used in cellular phenotypic characterization. (A) Normal human tonsil tissue consists of distinct tissue structure with a clear separation between the epithelial layer and B cells. (B) Human tonsil tumor exhibits a loss of structure organization and the epithelial layer. (C) Lymphoma of human tonsil is characterized by a complete absence of normal tissue structure and a disarray of B cells. This is an example ofhow multiplex tissue staining methods, as disclosed herein, can be used to identify tissue characteristics essential for distinguishing between cancerous and normal tissue.EXAMPLE 4: LABELING OF TISSUE WITH PRIMARY ANTIBODIES AND TYRAMIDES

[0142] Analyzing spatial relationships within organs and between cells on tissue can be used to investigate organization of biomolecules, cellular structures, cellular communication, and how their arrangement influences biological function and behavior. Factors to keep in mind when multiplexing include, for example: relative target abundance, antibody specificity, target localization, fluorescence output per dye, fluorophore photostability, and spectral separation of dyes.

[0143] Single tissue sample can be labeled using a combination of HRP -mediated signal amplification and primary antibody conjugates using the multiplex labeling methods disclosed herein. For example, the labeling process can include multiple rounds of labeling to achieve covalent fluorophore labeling on the sample using HRP -mediated signal amplification, ensuring no cross-reactivity with upcoming primary antibody labeling. Subsequently, a primary antibody labeling mix can be added in a single step.

[0144] In a separate experiment, a single tissue sample was labeled using multiplex , polyHRP labeling methods disclosed herein. The labeling process included multiple rounds of HRP - mediated amplification to achieve covalent fluorophore labeling on the sample.

[0145] An image of FFPE human small intestine tissue labeled without spectral unmixing using an EVOS M7000 Imaging System (Thermo Fisher Scientific) with a combination of 9 spatial dyes is shown in FIG. 10. The sample was stained using HRP -mediated amplification against primary antibody against E-cadherin (detected with Dye 488 dye seen in green), primary antibody against vimentin (detected with Dye 555 dye seen in red) and primary antibody against SMA (detected with Dye 750 dye seen in white) and counterstained with DAPI (seen in blue). The multiplex assay was used to identify smooth muscle, submucosa, crypt, and villi structures in the tissue. The image in FIG. 10 demonstrates that use of bright fluorescent dyes with polyHRP -mediated labeling can provide high-fidelity multiplexing for a variety of validated antibody clones to identify different tissue structures.

[0146] In yet another experiment, fluorescent images (FIG. 11) were collected for normal human tonsil tissue and non-Hodgkin lymphoma human tonsil tissue on the EVOS SI 000 Imaging System. The labeling process used multiple rounds of HRP -mediated labeling to achieve covalent fluorophore labeling on the sample, ensuring no cross-reactivity with upcoming primary antibody labeling; subsequently, the primary antibody labeling mixture was added in a single step. Image (A) of FIG. 11 shows normal human tonsil tissue treated using three successive rounds of tyramide dye deposition to achieve covalent fluorophore labeling on the sample, ensuring no cross-reactivity with upcoming primary antibody labeling. Primary antibody against CD4 detected with Dye 488 (seen in green); primary antibody against CD8a detected with Dye 555 (seen in yellow), and primary antibody against SMA detected with Dye 700, (seen in white) followed by treatment in a single step with a mixture including two labeled primary antibody conjugates (CD45RB with Alexa Fluor Plus 594 (seen in red) and FoxP3 with Alexa Fluor Plus 647 (seen in magenta). Image (B) of FIG. 11 shows Non-Hodgkin lymphoma human tonsil treated using two successive rounds of tyramide dye deposition; Primary antibody against CD8a detected with Dye 555 (seen in yellow) and primary antibody against SMA detected with Dye 700 (seen in magenta) followed by labeling with a mixture of three primary antibody conjugates: CD68 with Alexa Fluor Plus 488 (seen in green). CD45RB with Alexa Fluor Plus 594 (seen in red); and FoxP3 with Alexa Fluor Plus 647 (seen in white).EXAMPLE 5: FLUOROPHORE SELECTION

[0147] This example describes considerations and steps in selecting fluorophores for use in spatial imaging applications disclosed herein. Table 2 lists strategies for optimal reagent selection. FIG. 12 shows images for a variety of tissue types and targets that can be visualized in spatial biology applications using the selection methods disclosed herein.Table 2: Reagent Selection Strategies

[0148] Fluorophores for use in the methods disclosed herein can be selected according to the below protocol:1. Identify main target of interest2. Determine antigen expression level3. Save brightest fluorophores for dimmest markers• Use on the most important targets• Use on worst resolved targets• Low / unknown expression• Poor access to antigen4. Minimize spillover using known expression patterns• Space out co-expressed markers• Mutually exclusive markers in adjacent channels5. Plan for autofluorescence6. Avoid using dim or low expressing targets in channels with wide spectrums• The ability to resolve populations is a function of autofluorescence, background, and co-expressing markers.EXAMPLE 6: 9-PLEX SPATIAL PROTEOMICS WORKFLOW FOR SINGLE CELLQUANTIFICATION IN BREAST TISSUE

[0149] A 9-plex spatial amplification assay was used to process and stain human invasive ductal carcinoma of breast tissue. Formalin-fixed, paraffin-embedded human breast invasive ductal carcinoma tissue samples were obtained from BioChain Institute Inc. (Newark, CA). The slides then were processed using a Bond RXm (Leica Biosystems) and stained with primaryantibodies from Thermo Fisher Scientific (see, Table 3) and the spatial amplification reagents.Images were acquired and spectrally unmixed on the EVOS SI 000 from Thermo Fisher Scientific.Table 3: Staining Concentration for Primary Antibodies

[0150] Data analysis of the multiplex immunofluorescence stitched image was performed on the Indica Labs HALO (version 4.0.5107.318) software (Leica BioSystems). Manual annotation using the Magnetic Pen identified vimentin positive and vimentin negative regions. Cell detection and phenotyping was performed using the Indica Labs-HighPlex FL version 4.2.14, utilizing the Halo Al Nuclei Seg V2-FL classifier. Phenotypes were identified by positive expression of specified markers in Table 4.Table 4: Phenotypes and Specified Markers

[0151] By employing the EVOS SI 000 spatial imaging system, high-resolution images of an entire tissue section of breast tissue were successfully generated using a 20x objective (shown in image A of FIG. 15). The unique staining patterns of the 8 different biomarkers and DAPI were distinguished in different channels (shown in B of FIG. 15). These images were used in downstream analysis for precise examination of 8 immunologically relevant targets in context of their specific colocalizations and interactions.

[0152] The study indicated that immune cell populations colocalize with extracellular matrix vimentin. Cellular phenotyping enabled characterization of the 1.06 million cells identified in the 81mm2tissue section (FIG. 16). Referring to image (A), 25% of the cells were assigned as immune cells. 23% of the nonimmune cells were proliferating. The percentage of both myeloid (CD68+ cells) and lymphoid (CD3+, CD4+, CD8+ and CD20+ cells) subpopulations in specific region of the tissue were calculated (B), specifically within the extracellular matrix vimentin positive area (C). While proliferating PCNA+ cells are found in both regions, the immune cells are dominantly in the extracellular matrix vimentin neighborhoods of the cancer tissue.

[0153] Analysis plots derived from the imaging data from FIG. 16 are shown in FIG. 17. The percentage of both myeloid (CD68+ macrophage) and lymphoid (CD3+, CD4+, CD8+ and CD20+ cells, B, cytotoxic T, and helper T cell) subpopulations in specific region of the tissue (shown in image A of FIG. 16) were calculated, specifically within the extracellular matrix vimentin positive area (shown in image B of FIG. 16). While proliferating PCNA+ cells are found in both regions, the immune cells are dominantly in the extracellular matrix vimentin neighborhoods of the cancer tissue (shown in image C of FIG. 16). The analysis provided crucial insights into the peritumoral restriction of immune cell subpopulations within this section, and information that would not have been evident in a non-spatial, bulk phenotyping assay such as flow cytometry or single cell RNA sequencing.

[0154] The high-resolution images of whole tissue sections with 9 spectrally unmixed channels described in this example highlighted the synergy between the described spatial proteomic dye labeling workflows and the EVOS SI 000 system. Further, HALO analysissoftware facilitated single cell phenotyping of the tissue revealing immune cell colocalization with vimentin positive areas. The results point to the benefit of a spectrally unmixed 9-plex sample analysis (8 biomarkers and a nuclear counterstain) when reviewing the spatial relationships between immune cell types within the tumor microenvironment. Optimizing the spatial amplification reagents, purposely paired to a spatial imager, and combining downstream single cell analysis enables comprehensive immuno-characterization of complex tissues in a single sample.EXAMPLE 7: MULTIPLEX STAINING OF SMOOTH MUSCLE ACTIN INCLUDING NEAR-IR EMITTING DYE

[0155] In a first experiment, smooth muscle actin (SMA) on FFPE human intestine was stained with Aluora™ 750 dye (Thermo Fisher Scientific) and then subjected to 7 cycles of labeling and re-probing using an HRP -mediated signal amplification method with subsequent antigen retrieval steps, as disclosed herein, to provide a sample stained with 8 dyes. Aluora 750 dye is a near-IR emitting dye (emission maximum of about 783 nm) that includes a tyramide capable of binding covalently to available tyrosines on proteins in the sample when used in the instant signal amplification method. In a second experiment, SMA on FFPE human intestine was stained with 7 labeling and stripping cycles, followed by labeling with the Aluora 750 dye to provide a sample stained with the same 8 dyes. Both samples were imaged on an EVOS M7000 Imaging System at match exposure conditions.

[0156] FIG. 18 shows images of the FFPE human intestine tissue samples. The left panel (A) shows an image after staining of smooth muscle actin (SMA) on FFPE human intestine with Aluora1M750 dye. The Aluora 750 dye is excited by the 750 nm laser and emits at 783 nm and includes a tyramide that can bind covalently to the tissue when used in an HRP -mediated signal amplification method. The sample was first stained with the Aluora 750 dye and subjected to 7 cycles of stripping. The right panel (B) shows a spectral image after staining of SMA on FFPE human intestine, where the sample was subject to 7 stripping cycles and then stained with the Aluora 750 dye. A comparison of the images shown in panel (A) and panel (B) of FIG. 18 demonstrates that the sample first stained with Aluora 750 dye showed no significant signal dimming with repeated antibody stripping relative to the sample stained with Aluora 750 after prior multiplex staining.

[0157] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and Examples detail certain embodiments and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the embodiment may be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.EXAMPLE 8: SLIDE MOUNTED TISSUE SAMPLE

[0158] A tissue set for spectral imaging can include a full complement of slides necessary to perform spectral imaging using a spectral unmixing workflow. An exemplary tissue set includes: 1) a multiplex tissue slide stained with multiple fluorophores; 2) a single-color control tissue slide stained with each unique fluorophore individually; and 3) an unstained tissue slide for use as a tissue autofluorescence control. Each tissue set can use FFPE tissue samples to improve the shelf-stability and utility for the end-user.

[0159] In general, tissue slides that can be used in spectral imaging workflows with spectral unmixing can be prepared as follows. Tissue slices, typically FFPE murine kidney or human tonsil, are treated with various primary antibodies and stained with different fluorophores. The stained tissue slices, when mounted on a slide, can serve as controls as part of the calibration process in spatial imaging workflows using spectral unmixing. Although slide-mounted tissues, pre-stained with tissue markers, are available commercially (e.g. FluoCells™ Prepared Slide #3 and cyro-preserved mouse kidney section with Alexa Fluor™ 488 WGA, Alexa Fluor™ 568 Phalloidin, and DAPI, all available from Thermo Fisher Scientific), commercial slides include a limited number of spectrally resolved fluorophores and have been optimized for viewing in a standard fluorescent microscope. Pre-stained slides on the market currently are not adequate to meet the demands of more advanced multiplex, spectral imaging workflows.

[0160] A multiplex tissue slide was prepared from murine kidney tissue stained with nine (9) fluorophores using the Aluora™ Spatial Amplification System (Thermo Fisher Scientific). Antibodies were selectively paired with the different fluorophores used in the Aluora Spatial Amplification System to maximize the spectral unmixing quality of the panel. Design of the primary antibody panel considered the relative intensities of the fluorophores, coupled with the expression levels of the related haptens for each primary choice to balance signal intensitiesacross channels. Markers with a high degree of colocalization were avoided to be placed in spectrally adjacent channels to aide in the downstream assessment of unmixing results. Additionally, the order of biomarker staining within the Aluora Spatial Amplification iterative labeling process was optimized to provide the most robust signal for each antibody. For the murine kidney set of stained tissue slides, the resulting panel of biomarker and fluorophore pairings were as follows: CK8-Aluora 430 dye, AQP4-Aluora 488 dye, CK19-Aluora 514 dye, AQP2-Aluora 555 dye, MCM2-Aluora 594 dye, CK18-Aluora 647 dye, AQPl-Aluora 700 dye, SMA-Aluora 750 dye, and DAPI as the nuclear counterstain.

[0161] Slides were mounted with a coverslip, using a mounting solution. Any suitable mounting medium that has the requisite optical properties can be used to mount the sample onto the surface of the slide. For spectral imaging applications, it can be advantageous to use a mounting media that exhibits a refractive index (RI) of at least that matches that of the microscope slide. Ideally, the RI is equivalent to the refractive index of the microscope slide (e.g., about 1.47 to about 1.52) and should be chemically and optically stable, such that the calibration slides can be stored over time without degradation. A representative example of a mounting media that can be used to prepare a slide for spectral imaging calibration is a glycerolbased mounting media such as ProLong™ Glass Antifade Mountant from Thermo Fisher Scientific. Another example of a glycerol-based, aqueous mounting media for embedding labeled tissue samples that provides an appropriate RI for spectral imaging applications includes a methacrylamide-based polymer, such as poly(N-methyl methacrylamide), and about -10% glycerol, buffered to -pH 8.4 using Tris buffer.

Claims

CLAIMSWhat is claimed:

1. A method for preparing a biological sample, comprising: a) providing a biological sample covalently bound to two or more populations of tyramides, each population comprising a plurality of tyramides, and each tyramide in the plurality of tyramides is linked to a detectable label, wherein the detectable labels in each population are the same, and wherein the detectable labels in different populations are unique; and b) removing non-covalently bound components from the biological sample.

2. The method of claim 1, wherein the detectable labels are fluorophores or chromogens or combinations thereof.

3. The method of claim 1, wherein at least one population of tyramides comprise a detectable label that is a fluorophore that exhibits light with an emission maximum in the far-red or near-IR spectral region upon excitation at an appropriate wavelength of light.

4. A method for preparing a biological sample, comprising: a) providing a biological sample covalently bound to a population of tyramides, comprising a detectable label, wherein the detectable label is a fluorophore that emits light with an emission maximum in the far-red or near-IR spectral region upon excitation at an appropriate wavelength of light; and b) removing non-covalently bound components from the biological sample.

5. The method of any one of claims 1 -4, further comprising covalently binding a first population of tyramides to the biological sample, the biological sample comprising a first marker, in a first HRP-mediated signal amplification reaction under a first set of conditions, wherein the first populations of tyramides binds in a localized area to the biological sample in the vicinity of the first marker, to provide a biological sample labeled covalently to a first population of detectable labels.

6. The method of claim 5, further comprising performing a first antigen retrieval step to unmask a second marker on the tissue sample.

7. The method of claim 6, further comprising:(a) covalently binding a second population of tyramides to the biological sample, in a second HRP -mediated signal amplification reaction under the first set of conditions, wherein the second population of tyramides binds in a localized area to the biological sample in the vicinity of the second marker, wherein each tyramide in the second population comprises a second fluorophore, to provide a biological sample labeled covalently to a second population of fluorophore; and(b) removing non-covalently bound components remaining from the second HRP- mediated signal amplification reaction from the biological sample.

8. A method of labeling a biological sample, comprising: a. providing a biological sample comprising a plurality of markers; b. treating the biological sample with multiple iterations of a labeling cycle, represented as [I]N, wherein I is a labeling cycle and N is the number of labeling cycles, wherein N = 2-20, wherein the labeling cycle (I) comprises: i. covalently binding a plurality of populations of tyramides to the biological sample, in an HRP-mediated signal amplification reaction under a first set of conditions, wherein each population of tyramides binds in a localized area to the biological sample in the vicinity of a marker, wherein each tyramide in within a population comprises the same detectable label, and wherein each population of tyramides comprises a unique fluorophore, to provide a biological sample labeled covalently to a population of detectable labels; and ii. removing non-covalently bound components remaining from the HRP- mediated signal amplification reaction from the biological sample, to provide a biological sample labeled with multiple unique populations of detectable labels, wherein the number of unique populations of detectable labels corresponds to the number of labeling cycles (N).

9. The method of claim 8, wherein each iteration further comprises an antigen retrieval step prior to step (b)(i) to unmask the marker on the tissue sample.

10. The method of any one of the preceding claims, further comprising detecting the detectable labels, wherein the detectable labels are fluorophores.

11. The method of claim 10, wherein a first population of fluorophores emits light with an emission maximum greater than 710 nm and exhibits a first fluorescence signal intensity.

12. The method of claim 10, wherein a first population of fluorophores emits light with a first emission maximum between about 710 nm to about 850 nm.

13. The method of claim 11 or claim 12, wherein a second population of fluorophores emits light with a second emission maximum between about 680 nm to about 720 nm and exhibits a second fluorescence intensity.

14. The method of claim 13, wherein the first fluorescence intensity and the second fluorescence intensity differ by a factor of 5 or less.

15. The method of claim 13, wherein a first population of tyramides is used at a first concentration in a first cycle, and a second population of tyramides is used at a second concentration in a second cycle, such that the first and second fluorescence intensities differ by a factor of 5 or less.

16. The method of any one of the preceding claims, further comprising imaging the biological sample, wherein a first population of fluorophores and a second population of fluorophores are detected in two neighboring channels of a spectral imaging system, wherein the first population of fluorophores is detected in a first channel and the second population of fluorophores is detected in a second channel.

17. The method of any one of the preceding claims, further comprising imaging the sample to identify the locations the markers in the biological sample.

18. The method of any one of the preceding claims, wherein the HRP -mediated signal amplification reaction further comprises:(i) binding a primary antibody to a marker in the biological sample to provide a primary antibody treated sample;(ii) binding a secondary antibody, wherein the secondary antibody is attached to one or more horseradish peroxidase (HRP) molecules, to the primary antibody to provide an HRP treated sample; and(iii) treating the first treated sample with a detectable tyramide under conditions to initiate the signal amplification reaction.

19. A biological sample comprising two or more different markers, wherein each marker is labeled with two or more detectable tyramides, wherein each of the two or more detectable tyramides is covalently bound to the sample in a localized area to the biological sample in the vicinity of the two or more different markers, wherein each of the two or more detectable tyramides comprises a different fluorophore, respectively, wherein a first fluorophore emits light with an emission maximum between 350 nm and 710 nm and a first fluorescent signal intensity upon excitation at an appropriate wavelength of light, and a second fluorophores emits light with an emission maximum between 710 nm and 850 nm and a second fluorescence signal intensity, wherein the first fluorescence intensity and the second fluorescence intensity differ by a factor of 5 or less.

20. The biological sample of claim 19, wherein the sample is labeled with eight (8) or more unique detectable tyramides, comprising eight or more different fluorophores, respectively.

21. The biological sample of claim 19, wherein the sample is disposed on an imaging support.

22. The biological sample of claim 21, wherein the imaging support is a microscope slide, cuvette, well or dish.

23. The biological same of claim 21, wherein the biological sample is embedded in a mounting medium, wherein the mounting medium has a refractive index of 1.47 - 1.52.

24. A composition, comprising: a) a first population of tyramides at a first concentration, wherein the first population comprises a plurality of first fluorophores capable of emitting light with an emission maximum between 350 nm and 710 nm and a first fluorescent signal intensity; and b) second population of tyramides at a second concentration, wherein the second population comprises a plurality of second fluorophores capable of emitting light with an emission maximum between 710 nm and 850 nm and a second fluorescence signal intensity, wherein the first and second fluorescence intensity differ by a factor of 5 or less.

25. The composition of claim 24, wherein the concentration of the first population of tyramides is different from the second populations of tyramides.

26. A kit for labeling a biological sample, comprising: a) two or more populations of tyramides, wherein each population of tyramides comprises a plurality of unique fluorophores capable of emitting light with an emission maximum between about 350 nm and about 850 nm upon excitation at an appropriate wavelength of light, wherein at least one population of fluorophores emits light with an emission maximum in the far-red or near-IR spectral region; b) a poly-HRP- labeled detection reagent; c) an antigen retrieval reagent; and d) instructions for performing two or more cycles of HRP-mediation signal amplification to label the tissue sample with the two or more populations of tyramides.

27. An imaging system, comprising: an imaging device; and a biological sample of any one of the preceding claims mounted in the imaging device.

28. An imaging system, comprising: an imaging device, and a controller including an electronic processor and a non-transitory, computer readable medium, wherein the controller is configured to: receive a selection of one or more fluorescent channels for imaging a biological sample of any one of the preceding claims; capture, with the imaging device, a raw image of the sample; unmixing the raw image to generate an unmixed image.

29. The composition, sample, kit, system, or method of any one of the preceding claims, wherein the biological sample is a tissue, cell, cell organoid, cell spheroid, 3D cell culture, or a whole organism.

30. The composition, sample, kit, system, or method of any one of the preceding claims, wherein the detectable label is a dye selected from a cyanine-based dye, a hemi-cyanine-based dye, a rhodamine-based dye, a coumarin-based dye, a pyrene-based dye, an indacene-based dye (e.g., BODIPY), and an indole-based dye.

31. The composition, sample, kit, system, or method of any one of the preceding claims, wherein the indole-based dye is DAPI (4',6-diamidino-2-phenylindole).

32. The composition, sample, kit, system, or method of any one of the preceding claims, wherein the detectable label further comprises a water-solubilizing group.

33. The composition, sample, kit, or system, method of claim 32, wherein the water-solubilizing group is a poly(ethylene glycol) or sulfonate group.

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