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WO2025212919A2 - Compositions et procédés pour applications d'imagerie spatiale - Google Patents

Compositions et procédés pour applications d'imagerie spatiale

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Publication number
WO2025212919A2
WO2025212919A2 PCT/US2025/023000 US2025023000W WO2025212919A2 WO 2025212919 A2 WO2025212919 A2 WO 2025212919A2 US 2025023000 W US2025023000 W US 2025023000W WO 2025212919 A2 WO2025212919 A2 WO 2025212919A2
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WO
WIPO (PCT)
Prior art keywords
sample
biological sample
population
tyramides
tissue
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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PCT/US2025/023000
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English (en)
Other versions
WO2025212919A3 (fr
Inventor
Leticia MONTOYA
Nancie MOONEY
Scott Clarke
Ognjen Golub
Austin Harvey
Mary Kristina HAMILTON
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Life Technologies Corp
Original Assignee
Life Technologies Corp
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Application filed by Life Technologies Corp filed Critical Life Technologies Corp
Publication of WO2025212919A2 publication Critical patent/WO2025212919A2/fr
Publication of WO2025212919A3 publication Critical patent/WO2025212919A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels

Definitions

  • 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.
  • reagents and methods to facilitate multiplexed spatial phenotyping in tissue samples are also provided herein. 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).
  • a method for preparing a biological sample 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.
  • Each iteration can further include an antigen retrieval step prior to step (b)(i) to unmask the marker on the tissue sample.
  • the method can further include detecting fluorescence emission from the populations of fluorophores.
  • a first population of fluorophores emits light with an emission maximum greater than 710 nm and exhibits a first fluorescence signal intensity.
  • a first population of fluorophores can emit light with a first emission maximum between about 710 nm to about 850 nm.
  • 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.
  • the first fluorescence intensity and the second fluorescence intensity differ by a factor of 5 or less.
  • a biological sample 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 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.
  • 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.
  • a composition in yet another aspect, 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.
  • the concentration of the first population of tyramides is different from the concentration of the second population of tyramides.
  • an imaging system that includes an imaging device and a biological sample, as disclosed herein, mounted in the imaging device.
  • 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 biological sample, as disclosed herein; capture, with the imaging device, a raw image of the sample; and unmixing the raw image to generate an unmixed image.
  • 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.
  • FIG. 1 is a general schematic for a spatial biology workflow.
  • FIG. 2 is a schematic of a multiplex labeling workflow depicting covalent attachment of a labeling reagent.
  • 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.
  • 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).
  • 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.
  • FIG. 6 is a general staining workflow for IHC depicting labeling using a primary antibody conjugate mixture with an organic fluorophore.
  • FIG. 7 shows a set of images of tissues labeled with primary antibody conjugates. Primary antibody conjugates were confirmed and evaluated against secondary antibody IHC labeling. Primary antibody conjugates were specific and comparable to primary-secondary for the three different targets.
  • PanCK cytokeratin
  • CD68 macrophage marker labeled on normal human tonsil with Alexa FluorTM 488 Plus
  • FoxP3 transcriptional regulator labeled on normal human tonsil with Alexa FluorTM Plus 647.
  • FIG. 8 is an image of human tonsil tissue labeled with primary antibody conjugates against PanCK (AE1/AE3) Alexa FluorTM 700 (seen in red), CD20 (L26) Alexa FluorTM Plus 750 (seen in purple), CD68 (KPI) Alexa FluorTM 488 (seen in blue) and Ki67 (SolA15) eFluorTM 506 (seen in green).
  • 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) eFluorTM 506 (seen in green);
  • C Ki67 (SolA15) Alexa FluorTM 514 (seen in yellow);
  • D CD8a (C8/144B) Alexa FluorTM 594 (seen in red);
  • E SMA (1 A4) Alexa FluorTM 700 (seen in blue).
  • FIG. 10 is an image of FFPE human small intestine labeled with four dyes to demonstrate individual target validation.
  • 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.
  • 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 eFluorTM 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 FluorTM 488;
  • E CD8a, T-cell marker labeled on normal human tonsil tissue with Alexa FluorTM 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
  • FIG. 13A is a general spatial biology imaging workflow.
  • FIG. 13B is a diagram illustrating a cyclic labeling process for multiplex labeling of a tissue sample, as described herein.
  • 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.
  • FIG. 15A show an image of an 81mm 2 area 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.
  • 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).
  • 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 mm 2 area 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 81mm 2 tissue section shown in (A). 25% of the cells were assigned as immune cells, and 23% of the nonimmune cells were proliferating.
  • FIG. 17 shows analysis plots derived from the imaging data from FIG. 16.
  • 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 AluoraTM 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.
  • the term “a,” “an,” “the” and similar terms used in the context of the disclosure are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context.
  • “a,” “an,” or “the” means “one or more” unless otherwise specified.
  • the term “or” can be conjunctive or disjunctive.
  • the term “and/or” refers to both the conjunctive and disjunctive.
  • the term “substantially” means to a great or significant extent, but not completely.
  • 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.
  • 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.”
  • “about” can mean within 3 or more standard deviations, per the practice in the art.
  • 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.
  • an “analyte” or “antigen” as used herein refers to any substance recognized by an antibody, or another means of detection.
  • 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.
  • 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).
  • a “conjugate” or “conjugated,” as used herein refers to two or more moieties directly or indirectly coupled together.
  • 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).
  • linker a molecule or group of atoms positioned between two moieties.
  • 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.
  • 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.
  • 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 different specific binding molecules that specifically bind different targets to provide a multiplexed assay that can provide detection of the multiple targets in a sample.
  • detectable labels examples 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.
  • 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.
  • 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.
  • 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).
  • Fluorophore as used herein is a molecule that emits detectable electromagnetic radiation upon excitation with electro-magnetic radiation at one or more wavelengths.
  • fluorophores 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.
  • fluorophore and fluorescent dye may be used interchangeably herein.
  • Chrophor refers to a chemical compound that can, by chemical or other means, be converted into a chromophore.
  • chromophore 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.
  • exemplary chromogens include, but are not limited to, naphthols, aryl diazonium salts, and 1,3 -diketones.
  • chromogens examples include 3,3'-diaminobenzidine tetrahydrochloride (DAB) and the naphthol tyramide conjugates described in WO2024/145053A1, incorporated herein by reference in its entirety.
  • DAB 3,3'-diaminobenzidine tetrahydrochloride
  • WO2024/145053A1 incorporated herein by reference in its entirety.
  • 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.
  • a target or analyte can be contained in the interior of the sample.
  • 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.
  • 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 presence of multiple genes, proteins, or other molecules in the same tissue sample, while maintaining their spatial location.
  • 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 also refers to phenol-containing tyramide-like derivatives and analogues, such as the iFluor StyramideTM 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 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).
  • a detectable label such as a fluorophore, chromogen, or hapten (e.g., biotin or DIG).
  • 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.
  • a biological sample e.g., whole tissue sections or cells
  • detectable labels e.g., fluorescent dyes
  • immunostaining e.g., direct or indirect staining with primary or secondary antibodies conjugated to fluorophores
  • 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).
  • 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.
  • an imaging support e.g., a microscope slide
  • Samples are prepared to account for tissue type, tissue preservation components, tissue thickness, antigen localization, antigen abundance and autofluorescence characteristics.
  • 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).
  • EVOS S1000 Spatial Imaging System Thermo Fisher Scientific
  • analysis software such as the Indica Labs HALO analysis software.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a pixel mask may be defined as the pixels from the foreground channel used in averaging the intensity in all the channels.
  • 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.
  • 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.
  • 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, on a plurality of calibration samples, each calibration sample in the plurality of calibration samples stained with a different fluorophore included in the plurality of fluorophores.
  • SCC single color control
  • 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.
  • 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.
  • 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.
  • immunohistochemistry Assays, 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.
  • 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.
  • 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.
  • 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.
  • IHC systems include, but are not limited to, EnVisionTM (DakoCytomation), Powervision® (Immunovision, Springdale, AZ), the NBATM kit (Zymed Laboratories Inc., South San Francisco, CA), HistoFine® (Nichirei Corp, Tokyo, Japan).
  • 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.
  • 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 a plurality of unique detectable labels, as disclosed herein, such that the population can be detected by a spectral imaging system.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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).
  • a population of fluorophores can emit in the visible spectral region (e.g., 400 nm to 800 nm).
  • 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).
  • 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).
  • a set of fluorophores for multiplex spectral imaging applications can include fluorophores that fall within the channels of the 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.
  • 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.
  • Detectable labels useful for staining cells and tissues can be in the form of a conjugate.
  • 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.
  • a fluorophore can be covalently bound to a protein (e.g., antibody) to provide a fluorescently-labeled protein conjugate.
  • 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).
  • fluorophores can be conjugated (either directly or through a linker) to a substance that can covalently bind or associate with the biological sample.
  • 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).
  • 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.
  • the detectable label e.g., fluorophore, chromogen, hapten, and the like
  • a fluorophore can be covalently bonded (e.g., directly or indirectly linked) to a tyramine group to provide a fluorescently-labeled tyramide conjugate.
  • Detectable labels for biological applications can further include groups for improved water-solubility.
  • 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.
  • PEG poly(ethylene glycol)
  • 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.
  • reactive functional groups e.g., amine, carboxylic acid, azide, alkyne, succinimidyl ester, sulfonyl chloride, maleimide, and the like
  • reactive fluorophores include, but are not limited to: sulforhodamine 101 sulfonyl chloride (TexasRedTM or TexasRedTM sulfonyl chloride; 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);
  • 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.
  • fluorescent proteins such as green fluorescent protein (GFP) and its analogues or derivatives
  • fluorescent amino acids such as tyrosine and tryptophan and their analogues
  • fluorescent nucleosides 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
  • nucleosides fluorescent nucleosides
  • 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.
  • Thermo Fisher Scientific including, e.g., eFluorTM dyes, Alexa FluorTM dyes (e.g., Alexa FluorTM 350, 488, 555, 568, 594, 647, 680 and 750), Alexa FluorTM Plus dyes, NovaFluorTM dyes, Oregon Green 488, Pacific Blue (3-carboxy- 6,8-difluoro-7-hydroxycoumarin), and Rhodamine Green.
  • Alexa FluorTM dyes e.g., Alexa FluorTM 350, 488, 555, 568, 594, 647, 680 and 750
  • Alexa FluorTM Plus dyes e.g., Alexa FluorTM Plus dyes
  • NovaFluorTM dyes Oregon Green 488, Pacific Blue (3-carboxy- 6,8-difluoro-7-hydroxycoumarin
  • Rhodamine Green Rhodamine Green.
  • fluorophores and reactive versions thereof suitable for spatial imaging techniques 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).
  • 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., QdotTM semiconductor nanocrystals from Thermo Fisher Scientific).
  • Methods disclosed herein can implement fluorescently-labeled tyramide and tyramide-like reagents. Examples of commercially available fluorescently-labeled tyramide and tyramide-like reagents include CFTM dye-labeled tyramides from Biotium (Fremont,
  • iFluor® StyramideTM reagents e.g. iFluor 350, 488, 546, 555, 568, 594, 647, 60, 700, and 750 StyramideTM
  • OpalTM 520, 540, 570, 620, 650, and 690 reactive fluorophores from Akoya Biosciences
  • Alexa FluorTM tyramides and AluoraTM Spatial Dyes e.g., Aluora 430, 488, 514, 555, 594, 647, 700, and 750 Spatial Dyes
  • Thermo Fisher Scientific Wired, MA.
  • Methods disclosed herein also can implement colometric tyramide and tyramide-like reagents.
  • the SuperBoostTM EverRed and EverBlue Colorimetric HRP Kits from Thermo Fisher Scientific can be used with enzymatic amplification methods, such as disclosed herein.
  • tyramides can be synthesized from a wide variety of commercially-available, reactive versions of fluorophores reagents described herein.
  • 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
  • AAT Bioquest e.g., iFluor® reactive dyes, such as iFluor 350, 405, 430, 450, 488, 514, 532, 546, 555, 594, 560, 647, and 700).
  • 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.
  • 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 low abundant 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.
  • 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.
  • 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 single treatment 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.
  • 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.
  • HR horseradish peroxidase
  • FIG. 2 illustrates a single cycle of a representative labeling workflow including enzyme-mediated signal amplification.
  • 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).
  • 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.
  • 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.
  • 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 high density 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.
  • 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.
  • a biological sample is covalently bound to a detectable label.
  • the detectable label includes a fluorophore, chromogen, or hapten.
  • the detectable label can be bound to the sample using the enzyme- mediated amplification process.
  • 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.
  • 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.
  • 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 the availability of appropriate compounds and methods, especially for detection outside of the visible range of the electromagnetic spectrum (e.g., far-red to near-IR).
  • kits for labeling tissue samples with fluorophores using enzyme-mediated signal amplification methods, including the Tyramide SuperBoostTM Kits from Thermo Fisher Scientific, Tyramide Amplification Kits from Biotium, (Fremont, CA), and the OpalTM 6-Plex Detection Kits from Akoya Biosciences (Marlborough, MA).
  • Tyramide SuperBoostTM Kits from Thermo Fisher Scientific
  • Tyramide Amplification Kits from Biotium, (Fremont, CA)
  • OpalTM 6-Plex Detection Kits from Akoya Biosciences (Marlborough, MA).
  • the Opal TSA assay kit achieves multiplex labeling using a series of fluorescent dyes having emission maxima ranging from 480 nm to 690 nm. Labeling of an 8 th target 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.
  • 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.
  • 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.
  • each of the fluorescently-labeled tyramides are covalently bound to the available tyrosine groups in the vicinity of unique markers on the tissue.
  • labeling strategies that can be used to significantly expand the number of targets that can be imaged using modern spatial biology imaging techniques.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the fluorophores can exhibit fluorescence 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.
  • the concentrations of each set of fluorophores can be adjusted to control the relative fluorescence intensities between the sets of labels.
  • 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.
  • 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.
  • 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.
  • 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.
  • fluorophores can be selected based on the spectral profiles of each fluorophore, as well as their relative fluorescence intensities.
  • 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.
  • fraction bleed through can be minimized when fluorophores detectable in neighboring channels of a spectral imaging system exhibit similar fluorescence signal intensities.
  • 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.
  • a biological sample e.g., tissue or cell
  • HRP-mediated process as disclosed herein
  • 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.
  • 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.
  • a workflow 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.
  • workflows disclosed herein can be used for detection of 7, 8, or 9 unique markers in a tissue sample.
  • workflows disclosed herein can be used for detection of 8-20 unique markers in a tissue sample.
  • 9-15 unique markers can be detected in a tissue sample using workflows disclosed herein.
  • a biological sample can be produced using the instant methods that is labeled with two or more detectable tyramides.
  • 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.
  • each set of detectable tyramides includes a different fluorophore.
  • 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.
  • the fluorescence intensities of the two populations of fluorophores differ by a factor of 5 or less.
  • biological samples that are labeled with 2 or more unique detectable sets of tyramides, wherein each set includes a spectrally unique fluorophore.
  • the biological sample is labeled with eight or more sets of detectable tyramides, wherein each set includes a spectrally unique fluorophore.
  • 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.
  • 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.
  • Fluorophores for use in the methods disclosed herein can be selected according to the below protocol:
  • 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 primary antibodies from Thermo Fisher Scientific (see, Table 3) and the spatial amplification reagents.
  • FIG. 17 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.
  • EXAMPLE 7 MULTIPLEX STAINING OF SMOOTH MUSCLE ACTIN INCLUDING NEAR-IR EMITTING DYE
  • SMA smooth muscle actin
  • AluoraTM 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.
  • a multiplex tissue slide was prepared from murine kidney tissue stained with nine (9) fluorophores using the AluoraTM 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 intensities across 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.

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Abstract

La présente divulgation concerne de manière générale des procédés et des systèmes d'imagerie spatiale, ainsi que des compositions et des kits destinés à être utilisés dans de tels procédés. La divulgation concerne des réactifs et des procédés qui peuvent être utilisés pour une détection réussie faisant appel à des microscopes à champ lumineux et fluorescents et à des systèmes d'imagerie spectrale d'une large gamme de marqueurs protéiques dans de nombreux types d'échantillons biologiques.
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