Tissue Imaging Replicas

Abstract

A method for imaging a biological sample is disclosed, the method comprising: linking at least one affinity probe to the biological sample, wherein the affinity probe comprises at least one cleavable tag; contacting the probe-linked biological sample with a non-selective capture surface to link the at least one cleavable tag to the capture surface; and then cleaving the at least one cleavable tag from the affinity probe so as to transfer the at least one tag to the capture surface, thereby generating a replica of the biological sample.

Description

[Technical Field]

[0001] Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 418,379, filed October 21, 2022, the entire contents of which are incorporated herein by reference.

[0002] Technical Field FIELD OF THE DISCLOSURE

[0002] The present disclosure relates to methods for imaging biological samples by generating replicas of the biological sample and imaging or probing such replicas, and to the use of replicas for single cell analysis. [Background technology]

[0003] background

[0003] The present disclosure is generally directed to methods for imaging or probing biological samples (e.g., tissues, cell pellets, cell smears, etc.), and more particularly to such methods that can be used to generate replicas of a subject's biological sample that can be imaged or probed.

[0004]

[0004] Analysis of biological samples, for example, through imaging of the sample, is routinely employed not only for diagnostic purposes but also in medical / biological research. For example, such analysis of tissue sections may be used for quantitative detection of cellular protein markers. In many cases, highly multiplexed techniques are required to analyze biomolecules such as polynucleotide and polypeptide content of tissue sections or individual cells. Summary of the Invention [Problem to be solved by the invention]

[0005] However, conventional techniques for performing such analyses present several drawbacks. Immunohistochemistry (IHC)-based techniques typically do not allow for multiplexed analysis of a large number of proteins. For example, by employing techniques such as immunofluorescence (IHC), only one to four protein markers of interest can be analyzed at a time. On the other hand, imaging mass cytometry (IMC) TM (IMC TM ), electron microscopy and ion beam probing, and even some optical imaging techniques, can damage the tissue under study.

[0006]

[0006] In addition, variations in tissue samples and the substrate on which they are placed can adversely affect the function of some analytical techniques. For example, variations in the composition of the substrate on which the tissue sample is placed can affect the detection signal of secondary ion mass spectrometry (SIMS). In addition, analysis of the tissue section under investigation can result in contamination of the analytical equipment used to analyze the tissue section. For example, the tissue section may contain salts, which can contaminate the plasma sampling cone of an ICP-MS (inductively coupled plasma mass spectrometry) system. In addition, using ICP-MS to analyze large cells can be difficult because large cells may only be incompletely disintegrated in single-cell analysis. In laser ablation sampling, ablation characteristics can differ due to changes in the composition of different tissue parts, such as from the cytoplasm to the nucleus. This can result in reduced quality of imaging data. For example, small portions of a cell's nucleus may detach and land in different tissue spots, thereby creating "hot spots."

[0007]

[0007] Optical imaging techniques can also present some challenges: for example, in optical fluorescence techniques, tissue autofluorescence can interfere with the detection of probe tags attached to the tissue.

[0008]

[0008] Conventional optical methods for single cell analysis can also have some drawbacks. For example, clogging is one of the problems that occurs in cell analysis by flow cytometry and mass cytometry. Furthermore, in the analysis of cell smears, large cells can result in out-of-focus conditions for optical microscopy. [Means for solving the problem]

[0009] overview

[0009] In one aspect, a method for imaging a biological sample is disclosed, the method comprising: linking at least one affinity probe to the biological sample, wherein the at least one affinity probe comprises at least one cleavable tag, thereby creating a probe-linked biological sample; contacting the probe-linked biological sample with or bringing it into proximity with a capture surface to link the at least one cleavable tag to the capture surface; and cleaving the at least one cleavable tag from the affinity probe, thereby generating a replica of the biological sample.

[0010]

[0010] In practicing various embodiments, a variety of different types of biological samples can be employed. By way of example and not limitation, the biological sample can be any of a tissue section, cells, cell aggregates, cell smears, tissue biopsies, tissue microarrays, monolayers of adherent cells, one or more cells immobilized on a solid surface, and arrays of cells captured on a chip such as a SieveWell.

[0011]

[0011] The method can further include imaging a replica of the biological sample. In some embodiments, multiple different imaging modalities can be used to image the same replica. For example, a first image of the replica can be generated using a first imaging modality that causes no or minimal damage to the replica, and then a second imaging modality can be used to image the replica to generate a second image.

[0012] A variety of different imaging modalities can be employed, some examples of which include, but are not limited to, imaging mass cytometry, mass spectrometry, MALDI, secondary ion mass spectrometry, electron microscopy, fluorescence optical microscopy, and optical imaging.

[0013] In some embodiments, the capture surface has a thickness in the range of about 10 nm to about 1000 nm.

[0014] In some embodiments, the step of cleaving the cleavable tag is performed in a manner that substantially preserves information about the spatial distribution of the affinity probes in the biological sample. In some embodiments, the step of cleaving at least one cleavable tag is performed in a manner that substantially preserves information about the concentration of said affinity probes at each spatial location in the biological sample.

[0015] In some embodiments, at least one cleavable tag comprises a reporter element. By way of example and not limitation, the reporter element can be suitable for use in mass spectrometry, electron microscopy, fluorescence optical microscopy, MALDI, and SIMS, all by way of example. In some embodiments, the reporter element comprises a metal. In some embodiments, the reporter element comprises an imaging agent.

[0016]

[0016] In some embodiments, the materials include, but are not limited to, any of gels (such as ultrathin gels), metal-organic frameworks, covalent organic frameworks, nanomaterials, and porous silicon, among others.

[0017] In some embodiments, the capture surface can be a porous surface, such as a porous silicon surface. By way of example and not limitation, in some embodiments, such a porous surface can exhibit a porosity of at least about 10%. In some embodiments, the porous surface can be a porous organic film or nanomaterial. In some embodiments, the porous surface, such as a porous silicon surface, can be positioned on an underlying substrate, such as a silicon substrate.

[0018] In some embodiments, the capture surface is functionalized to have at least one capture moiety configured to capture a plurality of different tags (ie, different types of tags).

[0019] In some embodiments, the capture surface is substantially free of microbeads to which tags can be attached. In some embodiments, the capture surface is a selective capture surface, while in other embodiments, the capture surface is a non-selective capture surface.

[0020] In some embodiments, the capture surface is substantially free of the biological sample. In some embodiments, the capture surface is substantially free of any interference with the biological sample.

[0021]

[0021] In some embodiments, the step of linking at least one affinity probe to the biological sample includes staining the biological sample with a first affinity probe having a first cleavable tag to form a first replica of the biological sample, then removing any remaining affinity probes and associated cleavable tags from the biological sample, and following removal, staining the biological sample with a second affinity probe having a second cleavable tag to form a second replica of the biological sample.

[0022] In some embodiments, a first replica of a biological sample is generated using a first affinity probe having a first cleavable tag, and a second replica of the biological sample is generated using a second affinity probe having a second cleavable tag (i.e., a cleavable tag different from the first cleavable tag). The two replicas can be imaged using different imaging modalities, where each imaging modality is suitable for imaging one of the tags.

[0023] In some embodiments, a single replica of a biological sample is generated, where the replica comprises multiple different types of cleavable tags. In some embodiments, affinity probes with multiple different types of cleavable tags can be employed to generate the replica of a biological sample. In some embodiments, such replicas can be imaged using different imaging modalities, where each imaging modality is suitable for imaging one type of tag.

[0024]

[0024] In some embodiments, the method includes, following formation of the replica, removing at least a portion of the remaining affinity probes, if any, from the biological sample and generating another replica using the biological sample.

[0025]

[0025] In a related aspect, a method for imaging a biological sample is disclosed, the method comprising: linking at least one affinity probe to the biological sample, wherein the at least one affinity probe comprises at least one cleavable tag; and transferring the at least one cleavable tag from the biological sample to a non-selective capture surface, wherein the spatial distribution of the transferred at least one cleavable tag on the non-selective capture surface substantially retains the relative spatial distribution of the at least one affinity probe linked to the biological sample.

[0026]

[0026] A further understanding of various aspects of the present teachings can be obtained by reference to the following detailed description in conjunction with the associated drawings, which are briefly described below. [Brief explanation of the drawings]

[0027] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] 2 is a flow chart illustrating the various steps in one embodiment of a method according to the present invention for imaging a tissue sample. [Figure 2A]

[0028] 1 shows a schematic representation of a non-selective capture surface and a tissue section stained with an affinity probe having a cleavable affinity tag. [Figure 2B]

[0029] Schematic representation of a stained tissue section brought into proximity with a capture surface to facilitate transfer of tags from the tissue section to the capture surface. [Figure 2C]

[0030] 1 shows a schematic representation of a capture surface with a cleavable tag transferred thereto. [Figure 3]

[0031] 1 shows a schematic representation of a cleavable tag with an associated reporter. [Figure 4A]

[0032] 1 shows a schematic representation of a porous capture surface disposed on a porous substrate. [Figure 4B]

[0033] 1 shows a schematic representation of a cleavable tag captured by a porous capture surface. [Figure 5]

[0034] 1 shows a schematic representation of a plurality of stained cells placed on a substrate in contact with a capture surface to facilitate the formation of stained cell replicas. [Figure 6]

[0035] 1 shows a schematic representation of multiple cells arranged on a substrate having multiple channels that facilitate the transfer of cleavable tags to a capture surface. DETAILED DESCRIPTION OF THE INVENTION

[0028] Detailed Description

[0036] In imaging biological samples, it is typically desirable to image multiple target analytes (parameters) of a sample, such as a tissue section. The problem of detecting more parameters than the imaging method allows can traditionally be overcome by cyclic staining of the sample, such as with cyclic immunofluorescence (CyCIF) or CODEX. These methods rely on staining the same sample multiple times and are therefore incompatible with imaging methods that damage the sample. For example, the IMC imaging modality can simultaneously image 50 parameters associated with a biological sample. However, the imaging process destroys the sample. While fluorescence microscopy can typically image 4–6 parameters associated with a biological sample, this imaging can be performed in multiple cycles to read additional parameters, e.g., up to 100 parameters using 16–25 cycles. However, this method is inherently time-consuming (limited by the rate of staining) and can be subject to autofluorescence effects or repetitive staining damage from the tissue being imaged.

[0029]

[0037] The present invention is generally directed to a method for imaging a biological sample, which can generate a replica of a biological sample, e.g., a tissue section (including FFPE or frozen), and can be used to image a target analyte, such as a biomolecule of interest, in the sample. Biomolecules can be classified as proteins, oligonucleotides, lipids, carbohydrates, or small molecules, or combinations thereof. Alternatively, or in addition, biomolecules can be classified by their functionality. Biomolecules are not particularly limited and can have a variety of different molecular structures and functions. For example, an oligonucleotide can be a single-stranded DNA molecule, optionally a cDNA that hybridizes under stringent conditions to a target nucleic acid analyte (e.g., a sample nucleic acid biomolecule), or the oligonucleotide can be an aptamer. For example, a biomolecule can be an oligonucleotide that specifically hybridizes to a target oligonucleotide, such as a target mRNA present in the sample (e.g., hybridizes to a sample oligonucleotide). The hybridization can be a sequence of more than 8, more than 10, more than 15, or more than 20 nucleotides. As described above, in some embodiments, biomolecules can be classified by their functionality. For example, the biomolecule may be an affinity reagent, an antigen (eg, an analyte that is specifically bound by an affinity reagent), or an enzyme substrate.

[0030]

[0038] As will be discussed in more detail below, embodiments of the methods according to the present invention offer many advantages, for example, they allow for the acquisition of images of a biological sample while preserving the biological sample, and further allow for the use of different imaging modalities to image the replica.

[0031]

[0039] The terms "affinity probe" and "affinity reagent" or "probe" are used interchangeably herein to refer to a compound that can specifically bind to a target molecule, e.g., a biomolecule such as a protein or an oligonucleotide. Typically, an affinity probe forms a covalent bond with the target molecule.

[0032]

[0040] By way of example, an affinity reagent may be an antibody (e.g., or a fragment thereof), an aptamer, a receptor (e.g., or a portion thereof), or any other molecule, such as a biomolecule, that specifically binds to a target (e.g., an avidin, such as streptavidin, which specifically binds to biotin). For example, an affinity reagent can be associated with an antibody and used to detect and / or analyze the presence of its target antigen in a sample, such as the presence of a cytokine, a viral protein, a cancer biomarker, etc. By way of further illustration and without limitation, some examples of affinity probes can include, but are not limited to, sommers, DARPins, affimers, avimers, knottins, monobodies, and affinity clamps.

[0033]

[0041] As used herein, the term "cleavable affinity tag," or "cleavable tag," or "affinity tag," or "tag" refers to a compound that can be part of an affinity probe and can be cleaved from the remainder of the affinity probe, e.g., by the application of energy, to release the affinity tag. For example, the affinity tag can be a compound that can be attached to a portion of the affinity probe via a cleavable chemical bond, e.g., a covalent bond that can be cleaved by irradiation with ultraviolet light. In such embodiments, the combination of the affinity probe and the affinity tag can be referred to as an affinity probe having a cleavable affinity tag. Some examples of suitable affinity tags include, but are not limited to, tags suitable for use in various methodologies, such as optical imaging, IMC, MALDI, SIMS, electron microscopy, fluorescence, OES, and IHC. The tag can comprise any suitable structure, including polymer-based, beads, particles, nanoparticles, oligonucleotides, and molecular tags.

[0034]

[0042] By way of example, in some embodiments, the tag can be a chemical moiety that provides a distinguishable signal indicative of the presence of a target analyte or analyte complex, where the tag is associated with the analyte or analyte complex, for example, through linkage to an affinity product that recognizes the analyte or analyte complex. In some embodiments, the tag can include an element or isotope (or multiple copies thereof) that provides the distinguishable signal. The tag can include, for example, an element or an isotope of an element that is associated with the analyte or analyte complex and measured to determine the presence of the analyte. The tag can also include any distinguishable component (e.g., an element or isotope or multiple copies thereof), for example, provided on the surface or within the body of the particle or otherwise associated with the particle, that helps distinguish the particle from other particles. Further details regarding examples of tags suitable for use in various embodiments can be found, for example, in U.S. Pat. No. 10,436,698 B1 and / or WO 2020055813 A1, each of which is incorporated herein by reference in its entirety.

[0035]

[0043] As used herein, the term "capture surface" refers to a layer of material having a surface area that can be used to capture affinity tags. A "non-selective capture surface" refers to a capture surface that can be used to capture multiple different affinity tags or other molecules of interest. A non-selective capture surface can capture multiple tags, rather than just one or more predetermined tags. For example, a non-selective capture surface can be used to capture signaling chemicals excreted by shed cells. In another example, a non-selective capture surface can be used to capture metabolites produced when cells are exposed to pharmaceutical compounds, e.g., to screen how cells of the same type process different pharmaceutical compounds. For example, tags collected from the same cells can provide information about how each cell changes as a result of exposure to a particular pharmaceutical compound. A non-selective capture surface can capture all versions of the affinity tags used in an experiment, while simultaneously washing away salts and other potential contaminants. This behavior can therefore be considered selectivity in the broad sense, whereas a non-selective capture surface will capture tags indiscriminately and therefore be non-selective among different tags.

[0036]

[0044] The term "replica" as used herein refers to a capture surface onto which cleavable tags and / or native atoms / molecules available from a sample are transferred.

[0037]

[0045] The term "about" when used herein to adjust a numerical value is intended to indicate a variation of up to ±10% around that value.

[0038]

[0046] The term "substantially" as used herein is intended to indicate a deviation, if any, of up to 10% from perfect conditions or conditions.

[0039]

[0047] As used herein, the term "linked" when used to describe the relationship between two moieties, e.g., a biological sample and an affinity probe, indicates that a connection exists between these moieties, e.g., via a covalent bond, a van der Waals bond, or an ionic chemical bond. For example, an affinity probe can be linked to a surface protein marker of a cell, e.g., a cell in a tissue sample.

[0040]

[0048] Referring to the flowchart of FIG. 1 , in one embodiment of a method according to the present invention for imaging a biological sample, an affinity probe having a cleavable affinity tag, or alternatively a natural biological compound, is linked to the biological sample (e.g., a tissue section, one or more cells, etc.). The biological sample is then contacted with or brought into proximity with a non-selective capture surface, and the affinity tag is cleaved from the affinity probe so as to transfer the affinity tag to the non-selective capture surface, thereby forming a replica of the biological sample. In some embodiments, the affinity probe having the cleavable affinity tag is contacted with the capture surface via the affinity tag, and then the affinity probe is cleaved. In other embodiments, the affinity probe having the cleavable affinity tag is brought into proximity with the capture surface, and then the affinity probe is cleaved, and then the affinity tag contacts the capture surface. In some cases, the movement of the tag to the capture surface is facilitated by electrical forces, by gravity or centripetal forces, by diffusion, or by liquid flow. When natural biological compounds are used, the compounds are removed from the replica in a similar manner. The replica of the biological sample can then be imaged. As described below, in many embodiments, a variety of different imaging modalities can be employed to image the replica. Replicas of cell smears can also be used for single-cell analysis in suspension.

[0041]

[0049] As a further example, FIG. 2A schematically illustrates a support substrate 200 on which a tissue section 202 is disposed. The tissue section is stained with an affinity probe 204 that includes a cleavable affinity tag 206. More specifically, in this embodiment, the affinity tag is linked (bound) to the remainder of the affinity probe via a cleavable chemical bond 207, e.g., a covalent bond that can be broken by exposure to UV radiation. Various staining techniques can be employed in the practice of the present teachings. By way of example, the staining step can be in the form of immunostaining or DNA / RNA hybridization. Some examples of affinity probes are described above. In some embodiments, the cleavable bond can be activated by light (e.g., UV), chemically (e.g., enzymatically), electrochemically, or thermally. In some embodiments, molecules of interest can be extracted from biological samples by liquid extraction, electrically induced movement (e.g., electrophoresis), osmotic effects, or high gravity (e.g., centrifugal force), among others.

[0042]

[0050] Also shown schematically in Figure 2A is a non-selective capture surface 208 disposed on an underlying substrate 209. As described in more detail below, cleavable tags can be transferred from stained tissue sections to the capture surface, as seen in Figure 2B. The resulting effect is shown in Figure 2C, where all of the affinity tags 206 have been cleaved and attached to the substrate 209.

[0043]

[0051] Referring to FIG. 3 , the cleavable affinity tag 206 can include a reporter 206a that can be interrogated using one or more imaging modalities. Generally, various imaging modalities can be employed for detecting the cleavable tag (e.g., the cleavable tag reporter). By way of example, in some embodiments, the cleavable tag reporter can be a metal element such as osmium or a lanthanide. In some embodiments, the cleavable tag reporter can be a fluorescent dye or an imaging agent for electron microscopy. Alternatively, MRI imaging reagents, radioactive imaging reagents, X-ray analysis imaging reagents, or molecules suitable for detection by organic mass spectrometry, such as SIMS, MALDI, or ESI, can be used. Furthermore, organic mass spectrometry can simultaneously read the reporter molecule and the native molecule. However, imaging mass spectrometry only reads the native element and tag and may damage or destroy the native molecule in the process.

[0044]

[0052] As shown schematically in Figures 2A and 2B, the support substrate 200 can be brought into proximity with a non-selective capture surface 208 (referred to herein for simplicity as capture surface 208) such that the cleavable tag 206 associated with the affinity reagent contacts or is in proximity to the capture surface.

[0045]

[0053] The cleavable tag can then be cleaved and transferred to the capture surface, as shown schematically in Figure 2C. Tag cleavage can be achieved using a variety of different energy modalities. By way of example, tag cleavage can be achieved using photocleavage, chemical cleavage, electrochemical cleavage, among others.

[0046]

[0054] In some embodiments, transfer of the cleaved tag to the capture surface can be facilitated using electrical forces, e.g., in a manner similar to how electrical forces are employed in electrophoresis, or via diffusion, or via liquid flow, as described in more detail below. By way of example, in some embodiments in which the capture surface and the underlying substrate on which the capture surface is disposed are formed of a porous material, transfer of the cleaved tag can be achieved via the flow of liquid through a biological sample of interest (e.g., a tissue section) and then through the porous capture surface, followed by its outflow through the channels of the underlying porous substrate. In some embodiments, transfer of the cleavable tag to the capture surface can be facilitated via artificial gravity, as generated in a centrifuge, via magnetic force, or other suitable force.

[0047]

[0055] The capture surface can be any suitable surface capable of retaining tags transferred from a biological sample, which in this embodiment corresponds to a stained tissue section. As described above, the capture surface can enable capture of multiple different tags. Some examples of suitable capture surfaces include, but are not limited to, ultrathin gels, metal-organic frameworks, covalent organic frameworks, nanomaterials, and porous silicon, among others. The use of organic capture surfaces can be particularly advantageous in applications where ICP-MS is utilized to investigate the capture surface after transfer of cleavable tags to the capture surface, for example, by facilitating laser ablation similar to material disruption in an ICP ion source. By way of example, in some embodiments, the capture surface can be a covalent organic framework. As is known in the art, covalent organic frameworks are a type of crystalline porous organic polymer that exhibits permanent porosity and a highly ordered structure. Some examples of covalent organic frameworks and methods for synthesizing them are disclosed in the article entitled "Covalent Organic Frameworks: Design, Synthesis, and Function" by Geng et al., published in Chem Rev. 2020 Aug 26:120(16):8814-8933, which is incorporated herein by reference in its entirety.

[0048]

[0056] In some embodiments, the capture surface can be a porous surface. By way of example, FIG. 4A schematically illustrates a porous capture surface 400 supported on an underlying substrate 402. FIG. 4B schematically illustrates the capture surface 400 with a plurality of tags 206 attached thereto. Transfer of the tags 206 to the capture surface can be accomplished, for example, as described above with respect to FIGS. 2A, 2B, and 2C. In the embodiments of FIGS. 4A and 4B, the underlying substrate 402 is porous, as is the capture surface 400. In some embodiments, the porous capture surface 400 can exhibit a porosity ranging from about 10% to about 99%. In some embodiments, the porous capture surface can increase the upper dynamic range for tag capture via the capture surface by up to 1000 times. A relevant measure is the surface area of ​​a spot of a particular diameter on a porous surface compared to a flat surface. By making the surface porous, the available surface area can be increased by up to 1000 times. This ratio therefore roughly translates to a gain in the number of tags that can be captured compared to a flat, non-porous surface. By way of example, and not limitation, the porous capture surface 400 can be in the form of a porous silicon layer. By way of example, the porous silicon layer can be formed on a silicon substrate, for example, via chemical etching. In other embodiments, the porous capture surface 400 can be formed of a porous organic film (e.g., an organic framework).

[0049]

[0057] In some embodiments, the porosity of the capture surface 400 can facilitate capture and retention of cleaved tags. Additionally, in some embodiments, the combination of a porous capture surface and a porous underlying substrate can facilitate transfer of cleaved tags from the tissue section to the capture surface via the flow of a liquid (e.g., deionized water) through the tissue section and the porous capture surface. In some such embodiments, the tags can be retained within the pores of the porous capture surface, or the tags can be retained within channels of the substrate.

[0050]

[0058] 2A and 2B, in this embodiment, the capture surface is substantially free of moieties that exhibit specific binding for cleavable tags. By way of example, in embodiments, the capture surface may be substantially free of microbeads that have moieties that exhibit specific binding for a single tag (i.e., they are functionalized to capture a given tag). In other words, in such embodiments, indiscriminate capture of cleavable tags by the capture surface is achieved as a result of the material composition and / or shape of the capture surface.

[0051]

[0059] In other embodiments, the capture surface can include moieties capable of binding multiple cleavable tags. In some cases, the capture surface can include a single moiety capable of binding multiple cleavable tags, thereby facilitating retention of the cleavable tags by the capture surface. Some examples of such moieties include, but are not limited to, surface modifications of covalent organic frameworks, such as those discussed in the above-referenced paper by Geng et al., entitled "Covalent Organic Frameworks: Design, Synthesis, and Function."

[0052]

[0060] Imaging of the capture surface following transfer of the cleavable tag to the capture surface can be achieved using a variety of different imaging modalities suitable for the particular tag, some examples of which include, but are not limited to, fluorescence imaging, imaging mass cytometry, SIMS, and electron microscopy, among others.

[0053]

[0061] In some embodiments, a single type of tag is used to generate replicas. In some such embodiments, such tags can be interrogated (imaged) via different imaging modalities. For example, tags with osmium reporters can be imaged via both electron microscopy and IMC. Some lanthanide tags can be imaged via both fluorescent imaging and IMC.

[0054]

[0062] The ability to image tags via multiple different imaging modalities can provide several advantages. For example, if one imaging modality is found to potentially cause damage to the replica, another imaging modality that causes only minor or no damage can be used preferentially as the first method for imaging the replica. Alternatively, a given imaging modality can record only certain information from a subset of tags, while another imaging modality can record different information from a different subset of tags. For example, electron microscopy can be used to read osmium as a tag with high spatial resolution, while subsequent IMC can be used to read all metal tags, including osmium, as independent reporters, but with reduced spatial information.

[0055]

[0063] The material composition of the capture surface can be designed to facilitate the use of a particular imaging modality to image the replica. For example, the capture surface material can facilitate efficient ablation at a selected laser wavelength when ICP-MS is employed as the imaging modality. In some embodiments, the use of an organic capture surface can facilitate ablation and ionization when imaging the surface via MALDI, where the organic surface itself can function as a matrix, eliminating the need to apply a MALDI matrix to the sample.

[0056]

[0064] In some embodiments, a biological sample (e.g., a tissue section) of interest can be stained with an affinity reagent having a cleavable tag, where the cleavable tag can be activated for cleavage by multiple different methods. For example, one type of cleavable tag can be activated for cleavage using one type of energy modality, and another type of cleavable tag can be activated for cleavage using another type of energy modality. This allows multiple replicas to be generated from the same biological sample (e.g., a tissue section). Alternatively, a first replica can be created using a first tag set, and the tissue can be stained with a second tag set. The second set of tags can be cleaved, for example, using the same method, but in this case, the tissue will be contacted with a second capture surface. This creates a second replica. This process can be repeated three or more times using the same tag cleavage method (e.g., UV light cleavage).

[0057]

[0065] For example, a first replica can be formed through cleavage and transfer of one type of tag from a biological sample to a capture surface using one type of energy modality, and a second replica can be formed through cleavage and transfer of another type of tag from the biological sample to a different capture surface using a different type of energy modality. If the sample is not stained all at once, different energy modalities are not necessary. In some embodiments, if a biological sample is stained twice, a first replica is created after the first staining, and a second replica is created after the second staining. This process can be repeated based on the number of different tags linked to the biological sample. In this way, a biological sample (e.g., a tissue section) can be stained once but used to create two or more replicas of itself.

[0058]

[0066] The present invention is not limited to imaging tissue sections, but rather can be used to image a variety of biological samples. By way of example, and referring to FIG. 5 , the present invention can be employed to generate replicas of a plurality of cells 500 disposed on an underlying support substrate 502. As with the previous embodiment, the cells 500 can be stained with an affinity probe (not shown), such as the affinity probes described above, that have a cleavable tag, and the cleavable tag can be transferred to a non-selective capture surface 504 in the manner described above, e.g., by contacting or bringing the cells into proximity with the capture surface and causing cleavage of the tag, thereby generating a replica.

[0059]

[0067] FIG. 6 schematically illustrates an embodiment in which a plurality of cells 600 are disposed on a substrate 602 having two opposing surfaces 602a / 602b, with a plurality of channels 604 extending between two sets of openings 604a / 604b formed in the surfaces 602a / 602b, respectively. This is an example of tag probing rather than tag imaging. Each spot corresponds to a single cell and is recorded (probed) as a single record of many tags. Furthermore, the cells in this example can be maintained alive for further experiments. The cells 600 are disposed on the surface 602a so as to be substantially aligned with the openings 604a. The cells are stained with an affinity probe (not shown) having a cleavable affinity tag (not shown), as described above. A replica of the cells can be generated by contacting the surface 602b with a non-selective capture surface 606 disposed on the underlying substrate 607. The cleavable tag can then be cleaved by application of energy (such as UV radiation), for example, in the manner described above, and transferred through channel 604 to the capture surface.

[0060]

[0068] Embodiments of biological sample replicas according to the present teachings can provide several advantages. For example, but not limited to, replicas can be used to image biological samples without causing any damage to the biological sample. This allows, for example, imaging the same biological sample multiple times using multiple different staining and / or imaging modalities. By way of example, multiple cycles of an IMC imaging modality can be used to image 50 parameters (e.g., 50 target analytes of interest) in each cycle, and this cycle can be repeated four times using, for example, four replicas to image 200 parameters of the same biological sample (e.g., a tissue section).

[0061]

[0069] As described above, the present invention allows a biological sample to be imaged via multiple different imaging modalities. For example, different tags can be used to form two replicas of the biological sample, and each replica can be imaged using a different imaging modality suitable for the particular tag. In another embodiment, multiple different types of tags can be used to form replicas of the biological sample, where each tag can be imaged via a different imaging modality. In other words, a single replica can be formed using different types of tags, where each type of tag can be investigated (imaged) via a different imaging modality.

[0062]

[0070] In some embodiments, a biological sample can be stained with an affinity probe having a first cleavable tag, and the stained biological sample can be used to form a first replica, which can be imaged via a first imaging modality. The biological sample can then be washed (e.g., via washing, etc.) from the affinity probe and / or tag residue. The washed biological sample can then be re-stained with a different type of tag (which can be imaged using a different imaging modality) using the same type of affinity probe cleavably attached to the new tag, or a different affinity probe cleavably attached to the new tag, and a replica based on the re-stained biological sample can be formed. Each replica can be imaged using a different imaging modality suitable for imaging the different tag. In other embodiments, the affinity probe substantially loses affinity probe activity upon cleavage of the cleavable tag from the affinity probe (e.g., a biological or chemical trigger cleaves the cleavable tag and inactivates the affinity probe), such that the affinity probe residue is substantially removed from the biological sample.

[0063]

[0071] Additionally, the use of replicas can reduce contamination of imaging equipment. For example, as discussed above, tissues can contain salts and excess organic structures that can contaminate the plasma sampling cone of an ICP-MS system. In contrast, in embodiments, replicas can contain only molecules that can be digested by the plasma. This means that structural materials can be selected to be completely vaporized in the ICP ion source, even though cellular material may still occasionally reach and adhere to the interface cone.

[0064]

[0072] Additionally, replicas can be formed so that the capture surface is substantially uniform, thereby making the imaging process more predictable. For example, in some embodiments, the use of substantially uniform, thin replicas (e.g., replicas having thicknesses ranging from about 10 nm to about 1000 nm) can essentially eliminate the occurrence of "hot spots" that can occur when using laser ablation to image tissue sections, as described above.

[0065]

[0073] Additionally, the thickness of replicas produced in accordance with the present teachings can be much smaller than the thickness of the respective biological sample, e.g., tissue section, and the smaller the replica thickness, the less material that needs to be removed (e.g., by ablation or ion beam imaging).

[0066]

[0074] Furthermore, using replicas according to the present invention with optical imaging techniques can also offer several advantages. For example, it can eliminate the problem of autofluorescence interference in tag detection (which can vary for different tissue samples). Replicas do not exhibit autofluorescence that varies depending on the type of tissue from which the replica is formed. Furthermore, the autofluorescence of replicas can be controlled by the choice of material for the capture surface.

[0067]

[0075] Furthermore, in embodiments, the use of replicas to image biological samples can also solve some of the problems associated with single-cell analysis by flow cytometry and mass cytometry. For example, the use of replicas to image cell smears can eliminate the problem of system clogging that sometimes occurs when liquid samples are analyzed. Furthermore, the use of replicas according to the present invention can solve some of the problems associated with imaging or recording tag signals of large cells. As mentioned above, large cells can result in out-of-focus conditions in optical microscopy and incomplete collapse in ICP-MS-based analysis. In some embodiments, these problems associated with imaging large cells can be eliminated by generating thin replicas of the large cells. In some embodiments, replicas having the same thickness can be used to image large and small cells.

[0068]

[0076] Those skilled in the art will appreciate that various modifications can be made to the above-described embodiments without departing from the scope of the present teachings.

Claims

1. 1. A method of imaging a biological sample, comprising: Ligating at least one affinity probe to the biological sample, wherein the at least one affinity probe comprises at least one cleavable tag, thereby creating a probe-ligated biological sample; contacting the probe-linked biological sample with or proximate to a capture surface to link the at least one cleavable tag to the capture surface; cleaving the at least one cleavable tag from the affinity probe, thereby generating a replica of the biological sample; A method comprising:

2. 10. The method of claim 1, wherein the biological sample comprises any of a tissue section, a cell, a cell aggregate, a cell smear, a tissue biopsy, a tissue microarray, a monolayer of adherent cells, one or more cells fixed to a solid surface, and an array of cells captured on a chip.

3. The method of claim 1 , further comprising imaging the replica of the biological sample.

4. 4. The method of claim 3, wherein the step of imaging the replica of the biological sample comprises utilizing a plurality of different imaging modalities to image the replica.

5. 5. The method of claim 4, wherein the imaging modality comprises any of imaging mass cytometry, mass spectrometry, MALDI, secondary ion mass spectrometry, electron microscopy, fluorescence optical microscopy, SIMS, and optical imaging.

6. The method of claim 1 , wherein the capture surface has a thickness in the range of about 10 nm to about 1000 nm.

7. 10. The method of claim 1, wherein the step of cleaving the cleavable tag is performed in a manner that substantially preserves information about the spatial distribution of the affinity probes in the biological sample.

8. 3. The method of claim 2, wherein the step of cleaving the at least one cleavable tag is performed in a manner that substantially preserves information regarding the concentration of the affinity probe at each spatial location of the biological sample.

9. The method of claim 1 , wherein the at least one cleavable tag comprises a reporter element.

10. 10. The method of claim 9, wherein the reporter element is suitable for use in mass spectrometry, electron microscopy, fluorescence optical microscopy, MALDI and SIMS.

11. 10. The method of claim 9, wherein the reporter element comprises any of a metal and an imaging agent.

12. The method of claim 1 , wherein the capture surface comprises a porous surface.

13. The method of claim 12 , wherein the porous surface exhibits a porosity of at least about 10%.

14. The method of claim 12 , wherein the porous surface comprises a porous silicon surface.

15. The method of claim 14 , wherein the porous silicon surface is positioned over an underlying silicon substrate.

16. The method of claim 12 , wherein the porous surface comprises a porous organic film or a nanomaterial.

17. 10. The method of claim 1, wherein the capture surface comprises any of an ultrathin gel, a metal-organic framework, a covalent organic framework, a nanomaterial, and porous silicon.

18. The method of claim 1 , wherein the capture surface is functionalized to have at least one capture moiety configured to capture a plurality of different tags.

19. The method of claim 1 , wherein the capture surface is substantially free of microbeads to which the tag can be attached.

20. 20. The method of claim 19, wherein the capture surface is a selective capture surface.

21. The method of claim 1 , wherein the capture surface is substantially free of interference with the biological sample.

22. 2. The method of claim 1, wherein the step of linking the at least one affinity probe to the biological sample comprises: staining the biological sample with a first affinity probe having a first cleavable tag to form a first replica of the biological sample; removing at least a portion of the first affinity probe from the biological sample; and, following the removal, staining the biological sample with a second affinity probe having a second cleavable tag to form a second replica.

23. The method of claim 1 , wherein the capture surface is substantially free of the biological sample.

24. 2. The method of claim 1, wherein the at least one replica of the biological sample comprises a first replica generated using a first affinity probe having a first cleavable tag and a second replica generated using a second affinity probe having a second cleavable tag, and further comprising imaging the first replica using a first imaging modality and imaging the second replica using a different second imaging modality.

25. The method of claim 1 , wherein the at least one affinity probe comprises a plurality of different types of cleavable tags.

26. The method of claim 1 , wherein the at least one affinity probe comprises a plurality of affinity probes having different types of cleavable tags.

27. 27. The method of claim 26, further comprising imaging the replica using multiple imaging modalities, each imaging modality being used to image a different type of tag.

28. 10. The method of claim 1, further comprising, following formation of the replica, removing at least a portion of the remaining affinity probes, if any, from the biological sample and generating another replica using the biological sample.

29. 1. A method of imaging a biological sample, comprising: ligating at least one affinity probe to the biological sample, wherein the at least one affinity probe comprises at least one cleavable tag; transferring the at least one cleavable tag from the biological sample to a non-selective capture surface such that the spatial distribution of the transferred at least one cleavable tag on the non-selective capture surface substantially preserves the relative spatial distribution of the at least one affinity probe linked to the biological sample; A method comprising:

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