Determining region of interest in a tissue section

Abstract

Provided herein are methods of determining a region of interest (ROI) in a tissue section for imaging mass cytometry (IMC) that include (a) providing a reference tissue section, wherein the tissue section and the reference tissue section are corresponding tissue sections, and wherein the reference tissue section is stained with a staining reagent; (b) identifying a reference ROI in the reference tissue section; and (c) determining the ROI in the tissue section by image registration based on the position of the reference ROI identified in the reference tissue section.

Description

[0001] DETERMINING REGION OF INTEREST IN A TISSUE SECTION

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 63 / 541,568, filed on September 29, 2023, which is incorporated herein by reference in its entirety.

[0004] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under grant numbers CA268083 and CA275681, awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] TECHNICAL FIELD

[0007] The present disclosure relates to the field of imaging with image mass cytometry (IMC). In particular, it relates to determining a region of interest (ROI) in a tissue section for imaging mass cytometry, by using a reference tissue section.

[0008] BACKGROUND

[0009] Image mass cytometry (IMC) allows simultaneous detection of multiple antigens and nucleic acid sequences in formalin-fixed paraffin-embedded (FFPE) tissue sections, in frozen tissue sections, and in cultured cells using mass or metal isotopes conjugated antibodies. In conventional IMC techniques, the tissue is generally ablated using a laser with a 1 pm spot size over a selected region of interest (ROI). The aerosolized, atomized, and ionized tissue materials are then fed into a time-of-flight mass spectrometer for analysis of isotope abundance. The scanning speed of conventional IMC techniques is limited and imaging a field of view of ~mm2(i.e., 1 million spots) could take a substantial amount of time. Thus, practically only a small region of interest within the ~mm2area instead of the whole slide is captured in conventional IMC techniques. However, identifying the region of interest within the tissue section to be imaged with conventional IMC acquisition systems is highly challenging and time consuming. This is because the tissue sections used for conventional IMC techniques do not contain color reagent staining (e.g., H&E) to enhance the visible tissue and its architectures, and the built-in optical system within the instrument is not of sufficient resolution for precisely delineating the histological structures. Therefore, improved robust and effective methods are needed to identify / mark the region of interest in IMC sections for streamlining IMC imaging.

[0010] SUMMARY

[0011] Provided herein are methods of determining a region of interest (ROI) in a tissue section for imaging mass cytometry (IMC), the methods comprising (a) providing a reference tissue section, wherein the tissue section and the reference tissue section are corresponding tissue sections, and wherein the reference tissue section is stained with a staining reagent; (b) identifying a reference ROI in the reference tissue section; and (c) determining the ROI in the tissue section by image registration based on the position of the reference ROI identified in the reference tissue section.

[0012] In some embodiments, the tissue section is a formalin-fixed paraffin-embedded (FFPE) tissue section. In some embodiments, the tissue section is a frozen tissue section. In some embodiments, a thickness of the tissue section is about 5 pm. In some embodiments, the tissue section and the reference tissue section are less than 5 sections apart. In some embodiments, the tissue section and the reference tissue section are directly adjacent to each other.

[0013] In some embodiments, the staining of the reference tissue section comprises H&E staining, immunohistochemistry staining, immunofluorescence staining, antibody staining. Periodic acid Schiff (PAS) staining, Masson’s tri chrome staining, or any combinations thereof. In some embodiments, the staining reagent comprises hematoxylin and eosin, a fluorophore, antigen specific antibodies, Masson's trichrome stain, Periodic acid-Schiff stain, or any combinations thereof.

[0014] In some embodiments, the method further comprises analyzing the ROI of the tissue section by imaging mass cytometry. In some embodiments, the analyzing comprises detection of up to 50 analytes in the ROI of the biological sample. In some embodiments, an analyte comprises a polypeptide, a metal-based drug conjugate, a polysaccharide, an antibody conjugated molecule, or a nucleic acid. In some embodiments, an analyte comprises collagen, cytokeratins, E-cadherin, smooth muscle actin, podoplanin, vimentin, CD45, CD14, CD16, CD68, CD163, CD206, FOXP3, CD3, CD4, CD8, CD45RA, CD45RO, KI67, or HLADR.

[0015] 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 to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety’. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0016] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0017] BRIEF DESCRIPTION OF DRAWINGS

[0018] FIG. 1 shows an exemplary schematic of an imaging mass cytometry7(IMC) acquisition system to setup a scanning area to identify a region of interest (ROI).

[0019] FIG. 2 shows an exemplary schematic of a workflow of digitally marking the ROI location in a tissue section.

[0020] FIG. 3 shows an IMC image of vasculature in pancreas tissue acquired using the proposed workflow to digitally mark the region of interest location in a tissue section.

[0021] FIG. 4 show an IMC image of hair follicle in skin tissue section acquired used the proposed workflow.

[0022] DETAILED DESCRIPTION

[0023] Image mass cytometry (IMC) is a technique used to analyze tissue specimens from humans or other species that can generate highly multiplexed single-cell information at subcellular resolution. IMC uses metal-isotope tagged antibodies for simultaneous labeling and detection of multiple markers in formalin-fixed paraffin-embedded (FFPE) tissue sections, frozen tissue sections, and in cultured cells. IMC produces highly sensitive images of labeled markers as it minimizes the interference of autofluorescence from tissue or crosstalk between probes. IMC also can co-detect protein and RNA to provide deeper insight and integrated analysis in tissue. However, the imaging speed of conventional IMC techniques is limited and often only a very small region of interest within the whole slide is imaged. Moreover, since the section used for IMC analysis is not stained, it is very challenging to find the region of interest to be analyzed effectively.

[0024] Provided herein are methods to identify7regions of interest (ROI) in a tissue section by using a reference tissue section (e.g. H&E stained adjacent section), wherein the tissue section and the reference tissue section are corresponding tissue sections (e.g., corresponding on an axis perpendicular to the axes used to define a ROI, e.g., a z axis), and wherein the reference tissue section is stained with a staining reagent, identifying a reference ROI in the reference tissue section, and determining the ROI in the tissue section by image registration based on the position of the reference ROI identified in the reference tissue section.

[0025] Various non-limiting aspects of such methods are described herein and can be used in any combination without limitation.

[0026] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0027] As used herein, the term “about”, when used in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0028] As used herein, an “analyte” refers to any biological substance, structure, moiety, or component to be analyzed. In some embodiments, an analyte can be a nucleic acid analyte. In some embodiments, an analyte can be a non-nucleic acid analyte. For example, in some embodiments, a non-nucleic acid analyte can include, but is not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (e.g., N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, viral proteins (e.g., viral capsid, viral envelope, viral coat, viral accessory, viral glycoproteins, or viral spike), extracellular and intracellular proteins, antibodies, and antigen binding fragments. In some embodiments, an analyte can be a peptide or a protein, including antibodies and enzymes. In some embodiments, an analyte can be detected directly. In some embodiments, an analyte can be detected indirectly, such as through detection of an intermediate agent, for example, a secondary antibody.

[0029] As used herein, the term “biological sample” or “tissue section” refers to a tissue sample obtained from a subject for analysis using any of a variety of techniques including, but not limited to, biopsy, surgery, and laser capture microscopy (LCM), and generally includes cells and / or other biological material from the subject. In some embodiments, a tissue section can be obtained from a eukaryote, such as a patient derived organoid (PDO) or patient derived xenograft (PDX). In some embodiments, a biological sample can include one or more diseased cells. A diseased cell can have altered metabolic properties, gene expression, protein expression. and / or morphologic features. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders, and cancer.

[0030] As used herein, a ‘'subject’’ refers to an animal, such as a mammal (e.g., human or a non-human simian), or avian (e.g., bird), or other organism, such as a plant. In some embodiments, the subject is cat, a dog, a goat, a human, a non-human primate, a rodent (e.g., a mouse or a rat), a pig, or a sheep. Subjects from which tissue sections can be obtained can be healthy or asymptomatic individuals, individuals that have or are suspected of having a disease (e.g., cancer) or a pre-disposition to a disease, and / or individuals that are in need of therapy or suspected of needing therapy.

[0031] Methods of Determining a Region of Interest (ROI)

[0032] Provided herein are methods of determining a region of interest (ROI) in a tissue section for imaging mass cytometry (IMC) that include (a) providing a reference tissue section, wherein the tissue section and the reference tissue section are corresponding tissue sections, and wherein the reference tissue section is stained with a staining reagent; (b) identifying a reference ROI in the reference tissue section; and (c) determining the ROI in the tissue section by image registration based on the position of the reference ROI identified in the reference tissue section.

[0033] Imaging mass cytometry and tissue sections

[0034] Imaging mass cytometry is a high multiplexing imaging platform that is capable of simultaneously detecting and visualizing multiple different analytes (e.g.. antigens, proteins, nucleic acids, etc.). IMC can be used as a tool to study complex tissue morphology, wherein IMC can generate high-dimensional data at subcellular resolution by using metal-tagged antibodies to simultaneously stain and analyze multiple analytes of interest on a tissue section.

[0035] In some embodiments, a tissue section is provided by cutting a biological sample (e.g., a tissue sample, tissue block) into thin slices. In some embodiments, a tissue block is sectioned into thin adjacent, cross-sections, wherein consecutive tissue sections are provided. In some embodiments, a thickness of a tissue section is about 3 to 10 pm (e.g., about 3 to 9 pm, about 3 to 8 pm, about 3 to 7 pm, about 3 to 6 pm, about 3 to 5 pm, about 3 to 4 pm, about 4 to 10 pm, about 4 to 9 pm. about 4 to 8 pm, about 4 to 7 pm, about 4 to 6 pm. about 4 to 5 pm, about 5 to 10 pm, about 5 to 9 pm, about 5 to 8 pm, about 5 to 7 pm, about 5 to 6 pm, about 6 to 10 pm, about 6 to 9 pm, about 6 to 8 pm, about 6 to 7 pm, about 7 to 10 pm, about 7 to 9 pm, about 7 to 8 irn, about 8 to 10 pun, about 8 to 9 pun, or about 9 to 10 pun). In some embodiments, a thickness of the tissue section is about 5 pun.

[0036] In some embodiments, a tissue sample, is embedded in a matrix e.g., optimal cutting temperature (OCT) compound to facilitate the tissue sectioning. OCT compound is a formulation of clear, water-soluble glycols and resins, providing a solid matrix to encapsulate biological (e.g., tissue) specimens. In some embodiments, a tissue sample is embedded with paraffin prior to being sectioned. In some embodiments, the tissue sectioning is performed using cryosectioning.

[0037] In some embodiments, a tissue sample is fixed in a fixative including alcohol, for example methanol. In some embodiments, a tissue sample is fixed in acetone or an acetonemethanol mixture. In some embodiments, fixation is performed after sectioning. In some embodiments, a tissue sample is fixed with a fixative including an alcohol (e.g., methanol or an acetone-methanol mixture) after freezing and / or sectioning. In some embodiments, a tissue sample is flash-frozen, and then the tissue sample is sectioned and fixed (e.g., using methanol, acetone, or an acetone-methanol mixture). In some embodiments, when a tissue sample is frozen (e.g., flash frozen using liquid nitrogen and embedded in OCT) followed by sectioning and alcohol (e.g., methanol, acetone-methanol) fixation or acetone fixation, the tissue sample is referred to as “fresh frozen”. In some embodiments, fixation of a tissue sample e.g., using acetone and / or alcohol (e.g., methanol, acetone-methanol) is performed while the sample is mounted on a substrate (e.g.. glass slide).

[0038] In some embodiments, a tissue sample is fixed (e g., immediately after being harvested from a subject). In some embodiments, a fixative is preferably an aldehyde fixative, such as paraformaldehyde (PF A) or formalin. In some embodiments, a fixative induces crosslinks within a tissue sample. In some embodiments, after fixing (e.g., by formalin or PFA), a tissue sample is dehydrated via sucrose gradient. In some embodiments, a fixed tissue sample is treated with a sucrose gradient and then embedded in a matrix (e.g., OCT compound). In some embodiments, a fixed tissue sample is not treated with a sucrose gradient, but rather is embedded in a matrix (e.g., OCT compound) after fixation. In some embodiments, when a fixed frozen tissue sample is treated with a sucrose gradient, it can be rehydrated with an ethanol gradient. In some embodiments, a PFA or formalin fixed tissue sample, which can be optionally dehydrated via sucrose gradient and / or embedded in OCT compound, is then frozen e.g., for storage or shipment. In such instances, the tissue sample is referred to as “fixed frozen”. In some embodiments, a tissue section is a formalin-fixed paraffin-embedded (FFPE) tissue section. In some embodiments, a tissue section is a frozen tissue section. In some embodiments, when sectioning a tissue sample, the adjacent cross sections are maintained in consecutive order. In some embodiments, adjacent cross sections can be in non- consecutive order but relatively nearby '‘local” sections. In some embodiments, a “reference tissue section” refers to a second tissue section that corresponds to (e.g., corresponds to an axis perpendicular to the axes used to define a ROI, e.g., a z axis) a first tissue section (e.g., “analyte tissue section”), wherein the first tissue section is used for analysis by imaging mass cytometry. In some embodiments, an analyte tissue section and a reference tissue section are directly adjacent to each other. In some embodiments, an analyte tissue section and a reference tissue section are separated by one or more tissue sections. In some embodiments, an analyte tissue section and a reference tissue section are fewer than 5 sections (e.g., one section, two sections, three section, or four sections) apart. In some embodiments, an analyte tissue section and a reference tissue section can be up to 50 pm (e.g., up to 45 pm, up to 40 pm, up to 35 pm, up to 30 pm, up to 25 pm, up to 20 pm, up to 15 pm, up to 10 pm, up to 8 pm, up to 6 pm, up to 5 pm, up to 4 pm, up to 3 pm, up to 2 pm, or up to 1 pm) apart, regardless of the number of intervening tissue sections between them.

[0039] In some embodiments, a tissue section (e.g.. reference tissue section) is stained with a staining reagent. In some embodiments, the staining of a reference tissue section comprises H&E staining, immunohistochemistry staining, immunofluorescence staining, antibody staining, Periodic acid Schiff (PAS) staining, Masson's trichrome staining, or any combinations thereof. In some embodiments, a staining reagent comprises hematoxylin and eosin, a fluorophore, antigen specific antibodies, Masson’s trichrome stain, Periodic acid- Schiff stain, or any combinations thereof. Non-limiting examples of staining reagents can include histological stains (e.g.. hematoxylin and / or eosin) and immunological stains (e.g., fluorescent stains). In some embodiments, a tissue section can be stained using any number of staining reagents, including but not limited to, acridine orange, Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, propidium iodide, rhodamine, or safranin. In some instances, a tissue section can be stained using known staining techniques, including Can-Grunwald. Giemsa, hematoxylin and eosin (H&E), Jenner’s, Leishman, Masson’s tri chrome, Papanicolaou, Romanowsky, silver, Sudan, Wright’s, and / or Periodic Acid Schiff (PAS) staining techniques. PAS staining is ty pically performed after formalin or acetone fixation. In some embodiments, the staining includes the use of a detectable label selected from the group consisting of a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound, or a combination thereof.

[0040] Rezion of interest (ROD and detection of analytes

[0041] In some embodiments, a region of interest (ROI) is identified in a tissue section. In some embodiments, a reference ROI is identified in a reference tissue section, and a ROI in a tissue section (e.g., ‘‘analyte tissue section) is determined by image registration based on the position of the reference ROI identified in the reference tissue section. As used herein, a “region of interest” refers to a portion of a tissue section that is selected to be analyzed. In some embodiments, a ROI can include most commonly an area of about 0.5 to about 2.5 mm2(e.g., about 1.0 to about 2.5 mm2, about 1.5 to about 2.5 mm2, about 2.0 to about 2.5 mm2, about 0.5 to about 2.0 mm2, about 1.0 to about 2.0 mm2, about 1.5 to about 2.0 mm2, about 0.5 to about 1.5 mm2, about 1.0 to about 1.5 mm2, or about 0.5 to about 1.0 mm2) of a tissue section.

[0042] As used herein, “image registration” generally describes the process of transforming different sets of data into one coordinate system. In some embodiments, image registration can involve spatially transforming a source image (e.g., from a reference tissue section) to align with the target image (e.g., an analyte tissue section). For example, a digital image can be taken of each of a reference tissue section and an analyte tissue section for analysis, wherein one or more regions of interest are annotated within the reference tissue section either manually or digitally. The tissue sections of each of the adjacent slides are then individually aligned with the reference slide, and the region of interest (ROI) from the reference slide is transferred to each adjacent slide.

[0043] In some embodiments, a method provided herein further includes analyzing the ROI of the tissue section by imaging mass cytometry. In some embodiments, the analyzing comprises detection of up to 50 analytes in the ROI of the biological sample. As used herein, an “analyte” can include any biological substance, structure, moiety, or component to be analyzed. In some embodiments, the term “target” can similarly refer to an analyte of interest. In some embodiments, an analyte comprises a polypeptide, a metal-based drug conjugate, a polysaccharide, an antibody conjugated molecule, or a nucleic acid.

[0044] In some embodiments, analytes can be broadly classified into one of two groups: nucleic acid analytes, and non-nucleic acid analytes. Examples of non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N- linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, viral coat proteins, extracellular and intracellular proteins, antibodies, and antigen binding fragments. In some embodiments, an analyte can be an organelle (e.g., nuclei or mitochondria).

[0045] In some embodiments, an analyte can include a cell surface feature. For example, a cell surface feature that corresponds to an analyte can include, but is not limited to, a receptor, an antigen, a surface protein, a transmembrane protein, a cluster of differentiation protein, a protein channel, a protein pump, a carrier protein, a phospholipid, a glycoprotein, a glycolipid, a cell-cell interaction protein complex, an antigen-presenting complex, a major histocompatibility complex, an engineered T-cell receptor, a T-cell receptor, a B-cell receptor, a chimeric antigen receptor, an extracellular matrix protein, a posttranslational modification (e.g., phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation or lipidation) state of a cell surface protein, a gap junction, and an adherens junction.

[0046] In some embodiments, an analyte can be derived from a specific type of cell and / or a specific sub-cellular region. For example, an analyte can be derived from cytosol, from cell nuclei, from mitochondria, from microsomes, and more generally, from any other compartment, organelle, or portion of a cell. In some embodiments, permeabilizing agents that specifically target certain cell compartments and organelles can be used to selectively release analytes from cells for analysis.

[0047] In some embodiments, examples of nucleic acid analytes include DNA analytes such as genomic DNA, methylated DNA, specific methylated DNA sequences, fragmented DNA, mitochondrial DNA, in situ synthesized PCR products, and RNA / DNA hybrids. Examples of nucleic acid analytes also include RNA analytes such as various types of coding and noncoding RNA. Examples of the different types of RNA analytes include messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), and viral RNA.

[0048] In some embodiments, methods described herein can be used to analyze and / or detect up to 50 (e g., up to 5, up to 6. up to 7, up to 8, up to 9. up to 10, up to 15, up to 20, up to 25, up to 30, up to 35, up to 40, or up to 45) analytes in a region of interest within a tissue section. In some embodiments, an analyte comprises any antigen detected by a protein. In some embodiments, an antigen can include, but is not limited to, markers of stroma (e.g., collagen, smooth muscle actin, podoplanin, vimentin), epithelial cells (e.g., E-cadherin, cytokeratins), immune cells (e.g.. CD14, CD16, CD68, CD163, CD206, CD3. CD4. CD8. FOXP3, CD45RO CD45RA), or functional or signaling states (e.g., KI-67, cleaved caspase 3, phospho-STAT3). In some embodiments, an analyte can include collagen, cytokeratins, E-cadherin, smooth muscle actin, podoplanin, vimentin, CD45, CD14. CD16. CD68, CD163, CD206. FOXP3, CD3, CD4, CD8, CD45RA, CD45RO, KI67, or HLADR.

[0049] EXAMPLES

[0050] The disclosure is further described in the following examples, which do not limit the scope of the disclosure described in the claims.

[0051] Example 1 - Region of interest (ROI) under acquisition system of image mass cytometry (IMC)

[0052] Although IMC provides a promising tool for multiplex analysis of human tissue, the image acquisition speed of IMC is very' limited. IMC uses a laser with a 1-pm spot size to ablate the tissue and analyze the abundance of the tagged isotope from feeding aerosolized, atomized, and ionized tissue materials to a time-of-flight mass spectrometer. In some embodiments, the scanning speed of IMC in the mm2region of a tissue section (i.e., 1 million spots) could take a substantial amount of time. As a result, instead of the whole slide, only a small field of view from the whole slide with regions in the range of mm2area are in practice imaged with IMC as it can take days or weeks to scan the whole slide.

[0053] However, identifying the region of interest in the tissue sections with the current IMC acquisition system is highly challenging and time consuming. This is mainly because the tissue section can only be viewed at low contrast due to no-chromogen staining and with a limited field of view in the IMC acquisition system (FIG. 1).

[0054] To address this challenge, a robust and effective workflow has been established to identify / mark the region of interest in the IMC labeled sections for streamlining IMC imaging (FIG. 2). The workflow utilized the adjacent H&E-stained sections where tissue features are more prominent to robustly identify the region of inters to be analyzed. This ROI locations from adjacent H&E images is then marked in the digital images of the IMC labeled sections to be analyzed through image registration. Incorporating this ROI-marked tissue image in the IMC acquisition system allows users to scan IMC images at the targeted locations almost instantly, bypassing the time-consuming, subjective process of ROI identification directly in the acquisition system. This process also provides the adjacent H&E images of the IMC analyzed region to improve the interpretability' of the IMC data. The tissue sections used for the IMC analysis were labeled with the desired panels of the isotope tagged antibodies. The tissue section adjacent to the IMC section was then obtained and stained with H&E or other chromogenic staining. The high-resolution image of the H&E- stained section and non-chromogen stained IMC tissue sections can then be obtained using a slide scanner or camera attached microscope. In some embodiments, only the tissue regions were scanned with high resolution to minimize the size of tissue image data. A low resolution of IMC tissue sections was also obtained from a conventional document scanner where the whole slide was scanned. The region of interests (ROI) was first identified in the H&E images. Multiple locations of regions of interest can be identified in the single section if needed. The H&E image was then first computationally aligned to the high resolution non-chromogen stained IMC sections using the custom two steps image registration algorithm, the coarse registration and deformation map registration. This alignment model allowed for the direct estimation of the coordinates of the marked ROI locations in the unstained high resolution IMC sections from H&E images. To improve the image registration accuracy between H&E stained image and non-chromogen stained IMC sections, the H&E section image and non-chromogen- stained IMC section image were transformed to similar appearance first using a custom color deconvolution algorithm. The same procedure was then applied to non-stained high resolution and low-resolution images of the IMC section to obtain the locations of the marked locations of ROIs on the low-resolution image of IMC sections. The low-resolution images of IMC section from document scanner with the ROI marked region can then be fed into the IMC acquisition system. The low-resolution image of IMC section with ROI marks can be aligned with the acquisition systems following standardized protocols of the IMC instrument, and the locations of selected ROIs were then directly mapped in the acquisition systems, bypassing the challenging and difficult process to identify ROIs directly on the acquisition system.

[0055] This workflow has been successfully demonstrated in performing IMC imaging in a pancreas tissue section. A vasculature feature surrounded by the acini tissue of pancreas was identified in the adjacent H&E images. The described workflow was used to obtain the mapped corresponding ROI location in the low resolution image of whole IMC section which was then used to obtained the IMC images of these target ROI regions (FIG. 3). The workflow was also performed in a skin tissue section (FIG. 4). The hair follicle of the skin was identified in the adjacent H&E and the workflow was used to accurately obtain the corresponding IMC images of this targeted region in the adjacent IMC sections. The workflow also provides the H&E image corresponding to the IMC acquired field which can greatly help explain and validate the IMC image data. Overall, these results demonstrate that the proposed workflow can minimize the effort of a technician / operator in identifying the region of interest manually under the acquisition system and largely improve accuracy and robustness of the IMC image acquisition process while minimizing human bias.

Claims

WHAT IS CLAIMED IS:

1. A method of determining a region of interest (ROI) in a tissue section for imaging mass cytometry (IMC), the method comprising:(a) providing a reference tissue section, wherein the tissue section and the reference tissue section are corresponding tissue sections, and wherein the reference tissue section is stained with a staining reagent;(b) identifying a reference ROI in the reference tissue section; and(c) determining the ROI in the tissue section by image registration based on the position of the reference ROI identified in the reference tissue section.

2. The method of claim 1, wherein the tissue section is a formalin-fixed paraffin- embedded (FFPE) tissue section.

3. The method of claim 1 , wherein the tissue section is a frozen tissue section.

4. The method of any one of claims 1-3, wherein a thickness of the tissue section is about 5 pm.

5. The method of any one of claims 1-3, wherein the tissue section and the reference tissue section are less than 5 sections apart.

6. The method of claim 4, wherein the tissue section and the reference tissue section are directly adjacent to each other.

7. The method of any one of claims 1-3, wherein the staining of the reference tissue section comprises H&E staining, immunohistochemistry staining. immunofluorescence staining, antibody staining. Periodic acid Schiff (PAS) staining, Masson’s tri chrome staining, or any combinations thereof.

8. The method of claim 4, wherein the staining reagent comprises hematoxylin and eosin, a fluorophore. antigen specific antibodies, Masson’s trichrome stain. Periodic acid-Schiff stain, or any combinations thereof.

9. The method of any one of claims 1-5, further comprising analyzing the ROI of the tissue section by imaging mass cytometry.

10. The method of claim 6, wherein the analyzing comprises detection of up to 50 analy tes in the ROI of the biological sample.

11. The method of claim 7, wherein an analyte comprises a polypeptide, a metal-based drug conjugate, a polysaccharide, an antibody conjugated molecule, or a nucleic acid.

12. The method of claim 7, wherein an analyte comprises collagen, cytokeratins, E- cadherin. smooth muscle actin, podoplanin, vimentin, CD45, CD14, CD16. CD68, CD163, CD206, FOXP3, CD3, CD4, CD8, CD45RA, CD45RO, KI67, or HLADR.

Images