Multiple tissue imaging

Abstract

An apparatus is attached to a microscope slide to form an enclosed fluidic chamber with input and output ports, where a tissue or cell sample on the slide can be processed using techniques such as immunohistochemical staining. The reagent flow within the chamber can be laminar and highly uniform across the sample surface to achieve staining without gradients. The sample can be imaged before, during, or after any given processing step. Methods of staining and imaging a sample can be achieved through one or more rounds of labeling, label removal or erasure, and imaging. The sample can be imaged multiple times to achieve very high multiplexing levels. The sample can be placed on an imaging device at different times and removed from the imaging device for specific steps.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 861991, filed June 14, 2019, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to systems and methods for imaging biological samples. Background Technology

[0004] Immunohistochemistry (IHC) is used to detect molecular targets of interest in cells and tissues, utilizing antibodies to bind to specific targets of interest and labeling systems capable of detecting antibodies. A variety of optical labeling systems have been designed, including colored dyes and precipitates, fluorescent molecules, and various nanoparticles. IHC has significant value in researching, diagnosing, and guiding the treatment of various diseases.

[0005] To understand certain biological or medical processes, knowing the number or presence of multiple molecular species in each cell can be crucial. This is because the role or function of a given cell population is not easily determined by imaging any single molecular species.

[0006] Multiplex IHC technology has been developed that can detect or quantify more than one molecular target, including targets located in the same compartment of a given cell. Summary of the Invention

[0007] This disclosure features methods and systems for multiplex imaging of biological samples. For example, the method may include forming a closed chamber around the sample on a substrate such as a microscope slide. The chamber has a relatively small volume and reagents can be introduced into the chamber to perform multiple labeling and imaging cycles on the sample. In each cycle, the sample can be labeled with one or more probes, and one or more images of the probes can be obtained. The probes can then be removed, and additional probes can be bound to the sample and images. Each type of probe typically selectively binds to one type of component in the sample (e.g., one type of protein label), and the corresponding image of that type of probe produces quantitative information about the distribution and quantity of the component in the sample. By performing repeated cycles of labeling and imaging, a relatively large number (e.g., 20 or more, 40 or more, or even more) of different sample components can be imaged and quantified.

[0008] By enclosing the sample in a relatively small chamber, the number of probes and trapping agents used to detect sample components can be relatively small, and the duration of labeling and imaging cycles can be kept at a manageable level, which is an important consideration for highly multiplexed analysis. Furthermore, the relatively small chamber volume allows the probe and trapping agent composition to flow through the biological sample in a laminar flow manner, ensuring that non-uniform staining (e.g., due to eddies, gradients, and other non-laminar phenomena) is controlled or even eliminated. In addition, a consistent chamber volume and a very small dead volume (5 μL or less) allow precise amounts of the probe and trapping agent composition to be dispensed into the chamber, reducing waste and spillage. During sample processing, fluid can be moved over the sample or circulated back and forth within the enclosed chamber to agitate and enhance the interaction between the reagents in the fluid and components near the sample surface (e.g., targets).

[0009] Another significant advantage comes from the ability to disconnect the enclosed chamber (and the samples within) from different stations or locations within the processing system, or even from the system itself, for storage. Because the samples are enclosed within the chamber, the substrate supporting the samples and the chamber can be moved between different locations and into storage, where the samples are preserved. The samples can then be retrieved from storage after days, weeks, months, or even longer for further processing and / or imaging steps. Furthermore, components of the chamber can be removed, and different types of analysis (e.g., DNA and / or RNA extraction and sequencing) can be performed on the samples.

[0010] In one aspect, this disclosure features a method for analyzing a biological sample, the method comprising: obtaining a biological sample mounted on a first substrate; securing a second substrate to the first substrate to form a closed chamber on the first substrate, the biological sample being located within an internal volume of the closed chamber; and performing a plurality of imaging cycles, wherein each imaging cycle comprises: (a) binding a probe to the biological sample; (b) obtaining an image of the probe bound to the biological sample; and (c) removing at least a portion of the probe from the biological sample, wherein the thickness of the internal volume between the first and second substrates is 250 micrometers or less.

[0011] On another aspect, this disclosure features a method for analyzing biological samples, the method comprising: obtaining a biological sample mounted on a first substrate; fixing a second substrate to the first substrate to form a closed chamber on the first substrate, the biological sample being located within an internal volume of the closed chamber; and performing a plurality of imaging cycles, wherein each imaging cycle comprises: (a) binding a probe to the biological sample; (b) acquiring an image of the probe bound to the biological sample; and (c) removing at least a portion of the probe from the biological sample, wherein the first and second substrates are substantially transparent; and wherein the imaging window formed by the first and second substrates has a diameter of at least 500 mm. 2 Horizons.

[0012] In another aspect, this disclosure features a method for analyzing a biological sample, the method comprising: obtaining a biological sample mounted on a first substrate; securing a second substrate to the first substrate to form a closed chamber on the first substrate, the biological sample being located within an internal volume of the closed chamber; and performing a plurality of imaging cycles, wherein each imaging cycle comprises: (a) attaching a probe to the biological sample; (b) obtaining an image of the probe attached to the biological sample; and (c) removing at least a portion of the probe from the biological sample, wherein the first substrate is formed of glass and includes a first port defining a first channel extending through the first substrate connected to the internal volume and a second port defining a second channel extending through the first substrate connected to the internal volume; and wherein the closest distance between the first and second ports, measured in a plane of the second substrate, is 25 mm or greater.

[0013] In another aspect, this disclosure features a method for analyzing biological samples, the method comprising: obtaining a biological sample mounted on a slide; contacting the slide with a gasket including a hole, wherein the sample is located within the hole; and contacting the gasket with a window to enclose the sample within an internal volume defined by the slide, the gasket, and the window, wherein the window defines an observation area having a length of at least 25 mm and a width of at least 15 mm.

[0014] On the other hand, this disclosure features a system for analyzing biological samples, the system comprising a labeling station having a fluid device, a translation device, an imaging station having an image detector, and a controller connected to the fluid device, the translation device, and the image detector, wherein the controller is configured such that during system operation, the controller: (a) acquires a first biological sample located on a first substrate and enclosed in a first chamber formed by a first substrate, a second substrate, and a gasket located between the first and second substrates, and acquires a second biological sample located on a third substrate and enclosed in a second chamber formed by a third substrate, a fourth substrate, and a gasket located between the third and fourth substrates; (b) positions the first biological sample at the labeling station using the translation device and activates the fluid device to deliver a first labeling agent to the labeling station. (c) Positioning the first biological sample in the first chamber using a translation device at the imaging station and activating an image detector to obtain an image of the first labeler in the first biological sample; (d) Positioning the second biological sample in the labeling station using a translation device while the imaging station has the first biological sample, and activating a fluid device to deliver the second labeler to the second biological sample in the second chamber; (e) Removing the first biological sample from the imaging station; (f) Positioning the second biological sample in the imaging station and activating an image detector to obtain an image of the second labeler in the second biological sample; and (g) Positioning the first biological sample in the labeling station and activating a fluid device to remove at least a portion of the first labeler from the first biological sample and deliver a third labeler to the first biological sample.

[0015] Any embodiments of the methods and systems disclosed herein may include any one or more of the following features.

[0016] The first substrate can be formed of a substantially transparent material, such as glass or plastic. The first substrate can be a microscope slide. The second substrate can be a window.

[0017] The method may include positioning a gasket between first and second substrates. The gasket may be formed of an adhesive material.

[0018] The method may include, before immobilizing the second substrate to the first substrate: incubating the biological sample in acetone; removing residual acetone from the biological sample; and immobilizing the second substrate to the first substrate after removing the residual acetone. The method may also include, before incubating the biological sample in acetone: removing paraffin from the biological sample; and exposing the biological sample to an antigen retrieval agent.

[0019] The sample can be a fresh frozen sample. The sample can be a formalin-fixed paraffin-embedded sample.

[0020] This method may include applying a staining agent to a biological sample prior to performing multiple imaging cycles. The staining agent may include at least one selected from the group consisting of counterstaining agents, chromogenic staining agents, and immunofluorescent staining agents. The staining agent may include at least one of DAPI and Hoechst staining agents.

[0021] The second substrate may include a first port formed by a first hole extending through the thickness of the second substrate and a second port formed by a second hole extending through the thickness of the second substrate, wherein the composition is introduced into the internal volume through the first port. The minimum distance between the first and second ports, measured in the plane of the second substrate, may be 20 mm or greater.

[0022] During each imaging cycle, the probe can be bound to a biological sample by introducing a composition containing the probe through a first port and removing a component of the composition through a second port. The method may include binding a trapping agent to the biological sample by introducing a composition containing a trapping agent through the first port prior to performing multiple imaging cycles.

[0023] The capture agent may include a binder that selectively binds to a sample component and an oligonucleotide linked to the binder. The binder may include an antibody or an antibody fragment. The oligonucleotide may include at least 15 nucleic acids.

[0024] The composition may include multiple different types of trapping agents, each type having a different type of binder that selectively binds to different sample components, and oligonucleotides specific to each type of binder. The composition may include at least five different types of trapping agents (e.g., at least 20 different types of trapping agents). Each type of trapping agent can be bound to the biological sample by simultaneously introducing the composition containing each different type of trapping agent into the internal volume of a closed chamber.

[0025] The probe may include an oligonucleotide that is at least partially complementary to the oligonucleotide of the trapping agent, and a labeling agent linked to the oligonucleotide. The oligonucleotide of the probe may include at least 20 nucleic acids.

[0026] The labeling agent may include a fluorescent portion.

[0027] Binding probes to biological samples can include binding one or more different types of probes to biological samples, wherein each different type of probe includes a different type of oligonucleotide linked to a labeler specific to each different type of oligonucleotide.

[0028] One or more different types of probes may include at least three different types of probes. By introducing a composition containing one or more different types of probes into the internal volume of a closed chamber, one or more different types of probes can be simultaneously bound to a biological sample. One or more different types of probes can bind to corresponding different types of trapping agents in the biological sample. One or more different types of probes can hybridize with corresponding different types of trapping agents in the biological sample.

[0029] The oligonucleotide of the probe can hybridize with the oligonucleotide of the trapping agent, and removing at least a portion of the probe from the biological sample can include dehybridizing at least a portion of the probe from the trapping agent. The method can include dehybridizing at least a portion of the probe from the trapping agent by heating the biological sample and / or by introducing at least one dehybridizing agent into the internal volume of a sealed chamber. At least one dehybridizing agent can include a liquid-dissolving agent.

[0030] The thickness of the internal volume can be 100 micrometers or less. The internal volume can be 0.20 cm. 3 or smaller (e.g., 0.10cm) 3 (or smaller).

[0031] The method may include connecting a port connector to at least one of the first and second ports before introducing the composition into the internal volume of a sealed chamber. The port connector may include a first member having at least one fluid channel aligned with one of the first and second ports. Connecting the port connector to at least one of the first and second ports may include adhering the port connector to a surface of a second substrate. The port connector may include a second member, and connecting the port connector to at least one of the first and second ports may include connecting the first and second members of the port connector to secure the port connector in place relative to the second substrate. At least one of the first and second members may include at least one magnet, and the first and second members may be positioned such that the at least one magnet secures the first and second members in place relative to the second substrate.

[0032] Embodiments of the methods and systems may also include any other features disclosed herein, and may include any combination of features, including combinations of features described individually in conjunction with different embodiments, unless otherwise expressly stated.

[0033] Some embodiments described herein relate to a computer storage product having a non-volatile computer-readable medium (also referred to as a non-transitory processor-readable medium) having instructions or computer code on it for performing operations of various computer implementations. A computer-readable medium (or processor-readable medium) is non-transitory because it does not inherently include transiently propagating signals (e.g., propagating electromagnetic waves carrying information over a transmission medium such as space or cable). The medium and computer code (also referred to as code) may be designed and constructed for one or more specific purposes. Examples of non-transitory computer-readable media include, but are not limited to, magnetic storage media such as hard disks, floppy disks, and magnetic tapes; optical storage media such as optical discs / digital video discs (CD / DVD), optical disc read-only memories (CD-ROMs), and holographic devices; magneto-optical storage media such as optical discs; carrier signal processing modules; and hardware devices specifically configured to store and execute program code, such as application-specific integrated circuits (ASICs), programmable logic devices (PLDs), read-only memories (ROMs), and random access memories (RAMs). Other embodiments described herein relate to a computer program product that may include, for example, the instructions and / or computer code discussed herein.

[0034] Some of the embodiments and / or methods described herein can be implemented by software (executing on hardware), hardware, or a combination thereof. Hardware modules may include, for example, general-purpose processors, field-programmable gate arrays (FPGAs), and / or application-specific integrated circuits (ASICs). Software modules (executing on hardware) can be expressed in various software languages ​​(e.g., computer code), including C, C++, Java, etc. TMRuby, Visual Basic TM and / or other object-oriented, procedural, or other programming languages ​​and development tools. Examples of computer code include, but are not limited to, microcode or microinstructions, such as machine instructions generated by a compiler, code for generating network services, and files containing high-level instructions executed by a computer using an interpreter. For example, embodiments may be implemented using imperative programming languages ​​(e.g., C, Fortran, etc.), functional programming languages ​​(Haskell, Erlang, etc.), logic programming languages ​​(e.g., Prolog), object-oriented programming languages ​​(e.g., Java, C++), or other suitable programming languages ​​and / or development tools. Other examples of computer code include, but are not limited to, control signals, encryption code, and compression code.

[0035] 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 disclosure pertains. While methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the subject matter, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are illustrative only and not restrictive.

[0036] Details of one or more embodiments are set forth in the accompanying drawings and the following description. Other features and advantages will become apparent from the description, drawings, and claims. Attached Figure Description

[0037] Figure 1A This is a schematic diagram showing an exploded view of a chamber formed on a substrate supporting the sample.

[0038] Figure 1B This is a schematic diagram showing an example of a gasket used to form a chamber.

[0039] Figure 1C This is a schematic diagram illustrating an example of a port connector.

[0040] Figure 2 This is a flowchart illustrating example steps for labeling and imaging fresh frozen tissue samples.

[0041] Figure 3 This is a flowchart illustrating example steps for labeling and imaging formalin-fixed paraffin-embedded tissue samples.

[0042] Figure 4A This is an image of a tissue sample stained with DAPI.

[0043] Figure 4BThis is an image of a tissue sample stained with FITC.

[0044] Figure 4C This is an image of a tissue sample labeled with the CD68 probe.

[0045] Figure 4D This is an image of a tissue sample labeled with whole-cell keratin probes.

[0046] Figure 4E This is an image of a tissue sample labeled with an E-cadherin probe.

[0047] Figure 5 This is a schematic diagram of a system used to analyze biological samples.

[0048] Figure 6 This is a schematic diagram of the controller.

[0049] The same reference symbols in different figures represent the same elements. Detailed Implementation

[0050] introduction

[0051] One multiple staining technique is called SIMPLE, described by Glass et al. in J. Histochem. Cytochem. 57(10):899-905 (2009). It involves immunostaining a sample using 3-amino-9-ethylcarbazole (AEC) as a detection system, attaching a coverslip, imaging it with a bright-field microscope, removing it from the microscope, removing the coverslip, and destaining it with ethanol to remove any visible AEC. This sequence is repeated for each target of interest in the sample.

[0052] Lin et al. described a technique called t-CyCIF in eLife 2018, 7:e31657. It involves applying up to three antibodies to a sample, each directly conjugated to a fluorescent label, applying a counterstain such as Hoechst 33342, applying a coverslip, imaging on a fluorescence microscope, removing the sample from the microscope, removing the coverslip, and inactivating the dye by photobleaching the fluorophore with hydrogen peroxide. This sequence is repeated for each set of targets in the sample, so the overall measurement can include many targets. The fluorescent dye is directly conjugated to the antibody, except in the final round, where a second antibody is used to obtain amplification.

[0053] These two techniques are not easily automated, are time-consuming to complete, and require repeated application and removal of coverslips, carrying the associated risks of manual handling and sample damage. For at least these reasons, they are not easily deployed in environments where many samples are handled routinely, such as in research, clinical trials, or clinical care. However, they are flexible because individual histological and imaging steps can be performed using a variety of devices, depending on the available equipment and the measurement objectives of the sample involved.

[0054] Used for sample imaging Reagents, methods, and systems are available from Akoya Biosciences (Menlo Park, CA) and allow for multiplexed sample imaging. Specifically, multiple oligoconjugated antibodies bind to multiple targets of interest, each with a unique oligosequence. For each antibody, a label consisting of an oligoconjugated fluorescent dye designed to hybridize only with that antibody is employed. After incubation with all primary antibodies, a set of three labels is introduced with different dyes, which specifically hybridize with the selected antibody. The sample is then imaged, with each dye corresponding to a given primary antibody. Dehybridization is performed, and the labels are washed off. This cycle is repeated until all targets of interest have been imaged.

[0055] The system includes staining and imaging equipment. Samples are held in an open well in the glass bottom of an inverted microscope stage. Reagents are added to or removed from the chamber via automated jetting. Sample processing is typically not performed between cycles. Furthermore, primary antibody incubation is usually used only once, resulting in significantly less processing time compared to the SIMPLE or t-CyCIF methods. However, in typical experiments, neither the imaging nor staining equipment can be used for other purposes. Automation can be achieved using an open chamber instead of microscope slides, but a more standard sample format would be preferable. Various aspects of the methods, systems, and reagents related to the methods are described in the following U.S. patents, the entire contents of which are incorporated herein by reference: 10,370,698; 9,909,167; 10,017,808; 10,000,796, and 10,006,082.

[0056] In summary, there is currently no rapid sequential staining IHC multiplex technique where samples do not undergo repeated coverslip attachment and removal, and which can process samples provided in standard microscope slide formats. Nor is there any existing technique to achieve this in a fully automated manner using standard formats.

[0057] The methods and systems described herein image samples in a workflow involving multiple rounds of labeling, imaging, and label removal. The entire workflow can be performed semi-automatically or fully automatically. Furthermore, the workflow can be performed using samples mounted on standard microscope slides. In other words, the system can perform sample processing, imaging, and labeling, enabling the automation of labeling and delabeling of multiple samples, and imaging can be performed on a variety of manual or automated microscopes without sample reconfiguration or coverslip manipulation between steps or cycles.

[0058] Therefore, multiplex IHC with sequential staining can be performed at very high throughput, with equipment for staining and imaging functions operating at high utilization, and equipment for one function not idle while another function is in progress. The system and method may include or implement a modular sample cassette for imaging and processing samples on a microscope slide, the cassette incorporating a window through which the sample can be imaged and set separately from the sample surface to form a microfluidic chamber. This chamber may include ports for introducing and removing reagents from the sample, is easily maneuverable, and can be connected to or disconnected from a fluid source.

[0059] In some embodiments, the sample is a tissue section or tissue microarray placed on a microscope slide, and the window is a thin glass slide connected to the microscope slide by a peripheral seal that maintains a predetermined distance between the window and the microscope slide. This distance can be uniform within the region forming the microfluidic chamber and can be in the range of 40 to 250 micrometers. The system and method introduce and / or remove fluid reagents from the chamber formed by the window, the slide, and the seal. Fluid can be introduced into the microfluidic chamber through holes or orifices on the surface of the window, and a manifold connects the fluid from the inlet port to the orifice in the window. The manifold is preferably arranged at opposite ends of the window, with the sample between them. The chamber design and flow rate create laminar fluid flow over the sample.

[0060] The location and size of the holes or orifices can be designed to produce substantially similar flow rates across all areas of the sample. This facilitates uniform staining of the sample, which is generally valuable in quantitative analysis, particularly in analyses combined with amplification protocols such as hybridization or enzyme-catalyzed deposition. Furthermore, controlled flow is readily achievable to expose the sample to fresh reagent material at a predictable rate when the sample is depleted or used up. In some embodiments, the orifices at one end of the window form arcs with equal spacing between them, and all orifices are equidistant from the inlet ports of the associated manifold. This produces a uniform, thin layer of flow across the sample. The same arrangement can be used for two manifolds.

[0061] In some embodiments, the substrate holds multiple samples, each on its own microscope slide with a windowed microfluidic chamber, thus allowing for efficient processing of large numbers of samples in groups.

[0062] The port formed in the manifold allows fluid to be connected into or out of the chamber from a pipette tip or tubing, or both. Tubing (if present) may be combined with quick-connect fittings or other accessories for easy connection of the chamber to an automated fluid assembly and / or a manual pipette, or removal of the chamber from the connection to an automated fluid assembly and / or a manual pipette.

[0063] During sample processing in a closed chamber, reagents can remain in contact with the samples for extended periods, allowing for sample incubation. Samples undergoing incubation can be easily handled or moved without interfering with the ongoing incubation process.

[0064] The device of this invention can typically be used over a wide temperature range, generally from -4°C to 45°C, and can be used within any smaller temperature range of this range. The internal volume of the sealed chamber can be relatively small, for example, about 40 microliters for a 4×2 cm window with a window-to-slide spacing of about 50 micrometers. The dead volume within the sealed chamber can be 5 microliters or less, reducing reagent volume and cost. More importantly, by achieving a very small dead volume, the amount of a given reagent remaining in the chamber after the removal step is very small, ensuring that contamination between steps is largely eliminated.

[0065] Generally, the closed chambers described in this article are inexpensive to manufacture and easy to use, making them applicable in a wide range of settings, including academic research, translational research, experimentation, and clinical care.

[0066] The methods described in this article include multiple labeling and imaging of samples. Some methods involve multiple labeling and imaging of formalin-fixed paraffin-embedded samples on microscope slides. Other methods involve labeling and imaging of fresh frozen samples.

[0067] To form a closed chamber for the sample on a first substrate, such as a microscope slide, a second substrate (e.g., a window such as a coverslip) is attached to the first substrate at a selected distance and has holes or pores within it. Reagents are introduced, removed, and moved across the sample surface by guiding the flow of reagents through these holes or pores. These reagents are used to label components in the sample (e.g., molecular targets), and the labels are imaged through the window using a microscope. One or more labels are removed, erased, or both, and the cycle of labeling, imaging, and removal or erasure is repeated. In this way, multiple images of the sample are obtained. These images can be analyzed to obtain information about the sample or the organism from which the sample was extracted.

[0068] In some embodiments of this type, the sample is incubated with one or more primary antibodies before forming the fluid chamber by attaching a second substrate to the first substrate. In other embodiments, the primary antibody incubation is performed in the chamber after its formation.

[0069] A variety of methods can be used for sequential staining and multiplex imaging of biological samples. These are not limited to, but include bright-field labeling with 3-amino-9-ethylcolbazol (AEC) with ethanol erasure; direct labeling of antibodies conjugated with fluorescent dyes with hydrogen peroxide erasure; indirect immunofluorescence labeling of primary antibodies with the fluorescent label erased by catalase or by stripping primary, secondary, or both antibodies; labeling with oligoconjugated fluorescent dyes that hybridize to corresponding oligosequences conjugated with antibodies; and label removal by dehybridization.

[0070] This method may also include staining with histochemical dyes such as eosin, hematoxylin, 4',6-diamino-2-phenylindole (DAPI), periodic acid-Schiff (PAS), methylene blue, Hoechst stains (e.g., Hoechst 33342, Hoechst 34580, Hoechst 333258), and others, which are removed by ethanol or other solvents. These can provide information about the presence, location, and state of various cell compartments, which is useful in many cases and complements molecular IHC information.

[0071] This method can also include amplified fluorescence multiplexing. In one embodiment, a primary antibody is conjugated to a unique oligonucleotide sequence; labeling is performed using an enzyme catalyst, such as horseradish peroxidase (HRP) conjugated to the corresponding oligonucleotide sequence; a labeling substance is introduced into a device holding the sample and hybridizes with its corresponding antibody; tyramine signal amplification is used to deposit a fluorescent dye; and the label is dehybridized and washed away. This process can be repeated multiple times with different dyes having different spectra and the sample can be imaged. Optionally, the dye can be subsequently inactivated using hydrogen peroxide and light.

[0072] In some embodiments, rolling circle amplification (RCA) can be used to amplify probes in one or more labeling and imaging cycles, or linear and / or branched oligostructures can be used to attach multiple dye molecules to each labeled antibody.

[0073] Furthermore, any of the aforementioned methods can be combined. For example, sample processing and analysis methods may include one or more rounds of IHC labeling, imaging, and label removal or erasure, as well as one or more rounds of histochemical staining, imaging, and removal.

[0074] The sealed sample chamber can remain in place during multiple imaging and labeling cycles. This is advantageous because the workflow is practical for handling large volumes of samples or processing them in an automated manner. Furthermore, the samples are less likely to be damaged by processing steps such as attaching or removing the window.

[0075] In some embodiments, information about the sample obtained from a given labeling and imaging cycle can be used to select which targets to label and image in subsequent cycles. Therefore, image acquisition can be customized based on the sample, as information about the sample emerges during the process. The modular nature of sample processing makes it practical to isolate the sample after the IHC cycle for target selection is completed, until the sample components to be imaged in subsequent cycles have been selected, at which point sample processing resumes.

[0076] Sample box

[0077] As described above, the sample cassettes described herein are typically implemented by attaching one or more components to a substrate that supports the biological sample. A variety of different components can be used for this purpose. Figure 1A This is a schematic exploded view of components used to form an example sample cassette. A first substrate 150 supports a biological sample 151 (e.g., a tissue section). To form the cassette containing the sample 151, a gasket 102 is attached to the first substrate 150, and then a second substrate 101 is attached to the gasket 102. Securing the second substrate 101 to the first substrate 150 using the gasket 102 creates a sealed chamber with an internal volume, the thickness of which is determined by the thickness of the gasket 102. The field of view of the sealed chamber is effectively defined by the open area 113 of the gasket 102.

[0078] The second base 101 includes two or more ports to allow fluid to flow into and out of the internal volume of the chamber. Typically, each port is formed as an orifice extending through the second base 101 and connecting to the internal volume of the enclosed chamber. The second base 101 may have one or more inlet ports (e.g., two or more, three or more, four or more, five or more, or even more). Four inlet ports 103a-d are... Figure 1A The example is shown below. The second substrate 101 may also have one or more outlet ports (e.g., two or more, three or more, four or more, five or more, or even more). Four outlet ports 104a-d are shown in... Figure 1A The example is shown below.

[0079] An inlet manifold 105 may optionally be attached to a second substrate 101 via an adhesive layer 121 having orifices aligned with ports 103a-d. Fluid flowing into the inlet manifold 105 through inlet port 107 is conveyed through internal manifold channels and into ports 103a-d, thereby introducing fluid into the internal volume of the enclosed chamber. An outlet manifold 110 may optionally be attached to the second substrate 101 via an adhesive layer 123 having orifices aligned with ports 104a-d. Fluid flowing out of the internal volume of the enclosed chamber is conveyed through outlet ports 104a-d and into manifold 110, wherein fluid exits from exit port 112.

[0080] As described above, once assembled, the aforementioned components form a closed chamber around sample 151, which is located within the internal volume of the chamber. Reagents, compositions, and other fluids (as well as fluid-supported compounds) may be introduced into the internal volume to contact, react, bind, or otherwise interact with sample 151. Fluids, compositional components, and reagents may also be extracted from the internal volume, for example, by connecting a pump, vacuum source, or similar component to one or more outlet ports of the sample cartridge.

[0081] exist Figure 1A In this embodiment, the thickness of the internal volume of the sealed chamber—measured in a direction perpendicular to the sample support surface of the substrate 150—is determined by the thickness of the gasket 102. Alternatively, in some embodiments, the thickness of the internal volume of the sealed chamber is determined at least in part by other components of the chamber. In some embodiments, the thickness of the internal volume is 250 micrometers or less (e.g., 200 micrometers or less, 150 micrometers or less, 125 micrometers or less, 100 micrometers or less, 75 micrometers or less, 50 micrometers or less, 40 micrometers or less, 30 micrometers or less).

[0082] In particular, it has been observed that a thickness range between 30 and 250 micrometers promotes laminar fluid flow, ensuring close contact between fresh reagents and samples, thus reducing sample processing time compared to processing with more turbulent fluid flows. Furthermore, because the internal volume of the enclosed chamber is relatively small compared to the reagent volume used in benchtop processes and automated containers, reagent consumption is reduced compared to these techniques. Therefore, sample processing can be performed faster and / or more economically compared to these alternatives.

[0083] In some embodiments, the internal volume of the enclosed chamber is 0.40 cm³. 3 Or smaller (e.g., 0.35cm) 3 or smaller, 0.30cm 3 Or smaller, 0.25cm 3 or smaller, 0.20cm 3 Or smaller, 0.15cm 3 or smaller, 0.10cm 3 Or smaller, 0.08cm 3 Or smaller, 0.07cm 3 Or smaller, 0.06cm 3 Or smaller, 0.05cm 3 Or smaller, 0.04cm 3 Or smaller, 0.03cm 3 Or smaller, 0.02cm 3 Or smaller, 0.01cm 3 (or smaller).

[0084] exist Figure 1A In this configuration, one or both of substrates 150 and 101 may be substantially transparent. As used herein, “substantially transparent” means that less than 25% of the incident radiation is absorbed or reflected by the substrate at any wavelength from 450 nm to 650 nm. Because substrates 150 and 101 are substantially transparent, and manifolds 105 and 110 are located at the edge of substrate 101, the field of view of the enclosed chamber is determined by… Figure 1A The open area 113 of the center gasket 102 is defined. Alternatively, in some embodiments, other components used to form the enclosed chamber (such as one or more manifolds, base 101 and / or 150, and optional space filter layer) define the field of view of the enclosed chamber.

[0085] As used in this paper, the "field of view" of a closed chamber is the area of ​​its largest rectangular or square essentially transparent window that can image a sample on substrate 150. The field of view is determined by... Figure 1A The dashed rectangle 115 in the figure represents this. It should be noted that the field of view can be larger than the cross-sectional area of ​​a particular sample. In some embodiments, the field of view is at least 300 mm. 2 (e.g., at least 350mm) 2 At least 400mm 2 At least 450mm 2 At least 500mm 2 At least 550mm 2 At least 600mm 2 At least 650mm 2 At least 700mm 2 At least 750mm 2 ).

[0086] As used herein, the “observation area” of the enclosed chamber is the region through which a essentially transparent window allows imaging of a sample on substrate 150. The observation area can be equal to or larger than the field of view. The length of the observation area is its maximum dimension in the direction parallel to substrate 150, and the width of the observation area is its maximum dimension in the direction perpendicular to its length, with the width being less than the length. The corresponding length of the observation area (L) VA ) and width (W) VA )like Figure 1A As shown.

[0087] In some embodiments, the length of the observation area is 20 mm or greater (e.g., 25 mm or greater, 30 mm or greater, 35 mm or greater, 40 mm or greater, 45 mm or greater, 50 mm or greater, 55 mm or greater, 60 mm or greater, 65 mm or greater, 70 mm or greater). In some embodiments, the width of the observation area is 10 mm or greater (e.g., 15 mm or greater, 20 mm or greater, 25 mm or greater, 30 mm or greater).

[0088] The dimensions and positions of the substrate, gaskets, ports, and manifolds can be designed to generate laminar reagent flow over the sample 151 on the substrate 150. Therefore, the sample 151 can be labeled, imaged, and delabeled within the internal volume of a closed chamber. To ensure relatively uniform staining or labeling, the reagent flow to the sample 151 can be relatively similar at all locations on the sample surface. This is particularly important in processing that is highly dependent on reagent concentration or time, where reagents are either depleted due to interaction with the sample or typically do not reach saturation, instead producing kinetic-dependent results. An example of such processing is the IHC labeling amplification step.

[0089] Approximate homogeneity of the reagent or composition flowing through sample 151 can be achieved in a variety of ways. For example, in... Figure 1A In some embodiments shown, manifold 105 includes multiple flow channels coupled to multiple input ports 103a-d, and the impedance of the fluid flow along the fluid flow path defined by the flow channels and input ports is substantially the same. The input ports 103a-d are arranged in an arc within the enclosed chamber to aid impedance matching and deliver substantially equal flow rates through the ports. Due to the location and size of the chamber and ports, the flow is laminar at the flow rates used for sample processing. The fluid introduced as a separate flow at the input ports forms a continuous flow sheet within a few millimeters after entering the chamber. Therefore, the sample is processed uniformly with minimal reagent gradient.

[0090] In some embodiments, sample processing includes sequentially flowing a series of reagents over a sample. When a new reagent is introduced into the inlet port, fluid enters the chamber at a leading edge, the shape of which is determined by the arrangement of the ports. Here, the term "leading edge" is used to describe the transition of the chamber contents from one reagent to the next.

[0091] In this example, the leading edge forms an arc due to the arrangement of the input ports. With each new reagent introduced, this leading edge moves along the slide, traveling from one manifold to another. Typically, at least enough new reagent is introduced to ensure the leading edge has completely traveled through the sample area. Because laminar flow occurs within a closed chamber, the evacuation of previous reagents is rapid, and the number of fluid exchanges required to achieve a complete reagent flip is low compared to other slide-based sample processing methods. In some cases, only the advancement of the leading edge through the sample area is required. In other cases, several times the amount of new reagent is introduced, so that not even trace amounts of the earlier reagent remain in the sample area.

[0092] Generally, the second substrate 101 may include one or more inlet ports 103 (e.g., two or more, three or more, four or more, five or more, or even more). Additionally, the second substrate 101 may include one or more outlet ports 104 (e.g., two or more, three or more, four or more, five or more, or even more). Typically, the inlet ports 103 are located on a first side closer to the second substrate 101, and the outlet ports 104 are located on a second side closer to the second substrate 101 opposite to the first side. Typically, the inlet and outlet ports are located on opposite sides of the second substrate 101 such that fluid introduced through the inlet ports 103 and removed through the outlet ports 104 will flow across the surface of the sample 151 during transfer between the ports.

[0093] In some embodiments, such as Figure 1A As shown, the minimum or closest distance p between any inlet port 103 and any outlet port 104, measured in the plane of the second base. min The distance is 20 mm or greater (e.g., 25 mm or greater, 30 mm or greater, 35 mm or greater, 40 mm or greater, 45 mm or greater, 50 mm or greater, 55 mm or greater, 60 mm or greater, 65 mm or greater, or even greater). In the case where the second substrate 101 includes only two ports, the minimum distance is the closest distance between the two ports in the plane of the second substrate 101.

[0094] The first substrate 150 can be formed of various materials, including but not limited to various glass and / or plastic materials. For example, the first substrate 150 can be a microscope slide. The second substrate 101 can also be formed of various materials, including but not limited to various glass and / or plastic materials. As an example, the second substrate 101 can be a window, such as, but not limited to, a coverslip or another type of planar member. In some embodiments, the second substrate 101 is implemented as a 0.5 mm thick Corning Eagle XG glass (Corning, NY) measuring 24 × 55 mm.

[0095] The first substrate 150 may typically have a thickness of 1.1 mm or less (e.g., 1.0 mm or less, 0.9 mm or less, 0.8 mm or less, 0.7 mm or less, 0.6 mm or less, 0.5 mm or less, 0.4 mm or less, 0.3 mm or less, 0.2 mm or less, 0.1 mm or less). The second substrate 101 may typically have a thickness of 1.1 mm or less (e.g., 1.0 mm or less, 0.9 mm or less, 0.8 mm or less, 0.7 mm or less, 0.6 mm or less, 0.5 mm or less, 0.4 mm or less, 0.3 mm or less, 0.25 mm or less, 0.2 mm or less, 0.1 mm or less).

[0096] Typically, manifolds 105 and 110 can be formed from a variety of materials, including plastics, rubber-based materials, and metals. For example, in some embodiments, manifolds 105 and / or 110 are formed from polyetheretherketone (PEEK), which is solid-printed to provide ports for connecting them and internal conduits.

[0097] Gasket 102 is typically implemented as a pressure-sensitive adhesive, chosen for its good adhesion and chemical compatibility with the reagents, cleaning agents, and other fluids used in the process. In embodiments where the gasket thickness determines the thickness of the chamber's internal volume, the selection of the gasket provides a method for selecting the chamber thickness to a desired value. In some embodiments, for example, gasket 102 may be formed of an acrylic material, such as 3M Adhesive Transfer Tape 966 (3M, St. Paul, MN), although other materials may be preferred in other cases depending on the desired thickness and the chemistry of the reagents used in the process.

[0098] Adhesive layers 121 and / or 123 can typically correspond to pressure-sensitive adhesives as described above. Alternatively, epoxy resins or other adhesives, such as Master Bond EP21LV (Hackensack, NJ), can be used. Generally, the adhesive layers provide a liquid-tight seal between the manifold and the second substrate without interfering with fluid flow. Selection criteria include chemical compatibility with reagents used in sample processing and their adhesion to glass and associated manifold surfaces.

[0099] Other techniques can also be used to generate uniform flow with different geometries. For example, manifolds can have channels of different lengths, with diameters chosen to achieve approximately equal flow rates / volumes at the inlet ports.

[0100] For clarity, the terms input port and output port are used above, and fluid flow can be entirely or primarily in one direction. However, if it is advantageous for a particular sample processing operation, the enclosed chamber can be operated with fluid flow in either direction. For example, the flow can circulate in either direction to achieve an effect similar to that achieved by shaking and stirring during bench processing, i.e., promoting interaction between the sample and reagents. Furthermore, by circulating which reagent volume interacts with which sample area, this effect reduces the extent to which local sample reactions deplete the reagents in their vicinity.

[0101] exist Figure 1A In the middle, manifolds 105 and 110 provide a single orifice or port for fluid inlet and outlet. This can be coupled to the conduit by pressing a short section of #19 gauge stainless steel hypodermic material into the port and attaching the conduit to the protruding portion of the material using a friction fit with a suitably sized conduit fitting. If desired, the conduit can include a quick-connect fitting at the other end.

[0102] Alternative connection schemes are possible, in which manifolds 105 and / or 110 incorporate resilient seals such as O-rings, allowing the pipette to directly contact the inlet and / or outlet ports 107 and / or 112. This can be advantageous in situations where reagents are limited or expensive, as it minimizes the dead volume. Because the required volume of the filling chamber can be very small, the overall arrangement is highly efficient in terms of reagent use. In some embodiments, for example, the total dead volume within the closed chamber is 5 μL or less (e.g., 4 μL or less, 3 μL or less, 2 μL or less, 1 μL or less).

[0103] Various connections between the manifold and external fluid equipment are possible, allowing the same manifold to be connected to pipettes or pressure-fitting piping fittings at different times. In this way, the same enclosed chamber can be easily connected to manual fluid items, such as pipettes, or to automated fluid equipment via piping or other means.

[0104] An example of a closed room is as follows Figure 1A As shown. However, the enclosed chamber described here can be implemented in a variety of ways. Figure 1B A top view of another example of a closed chamber formed by a first base 150, a gasket 102, and a second base 101 is shown (for clarity, the first base 150 is not shown). Figure 1B (As shown in the image). Figure 1BAs shown, the chamber includes a single input port 103 and a single output port 104, each formed as a hole in the second substrate 101 extending through the thickness of the substrate. The opening region 113 of the gasket 102 is formed as an irregular hexagon (i.e., a hexagon with uneven side lengths). Ports 103 and 104 are positioned at opposite vertices of the hexagonal opening along the central axis of the gasket. Figure 1B The washer 102 can have the above-mentioned bonding Figure 1A The gasket 102 discusses any one or more properties.

[0105] Manifolds 105 and 107 can also be referred to as port connectors, particularly in cases where the enclosed space includes a single input port and / or a single output port. Other implementations of port connectors besides manifolds 105 and 107 may also be used. For example, Figure 1C A schematic diagram of another example of a port connector 160 is shown. The port connector 160 includes a first component 170 and a second component 172. A magnet 176 is embedded within the body of the first component 170 (optionally, as an alternative to or supplement to the first component 170, the magnet may be embedded within the body of the second component 172). Component 172 is formed of a magnetic material such as stainless steel. The first component 170 includes a connection to port 103 or port 104 (…). Figure 1B The hole 174 is located within the first member 170. The hole 174 is connected to the port 178 via an internal channel, and the port 178 is in turn connected to the fluid conduit 180.

[0106] To engage the port connector 160 with the second base 101, a first member 170 is located on top of the second base 101, with a hole 174 aligned with port 103 or 104. A second member 172 is positioned opposite the first member 170 on the underside of the first base 150. Magnetic force between the magnet 176 and the second member 172 ensures that the port connector 160 remains firmly in place.

[0107] Various other locking mechanisms can also be used to secure components 170 and 172 to the second base 101. For example, in some embodiments, mechanisms such as hooks, latches, pins, tabs, and keys can be used to lock components 170 and 172 in place relative to each other and relative to the second base 101. Alternatively, components 170 and / or 172 can be secured relative to the second base 101 using an adhesive, such as any of the aforementioned adhesives. When using an adhesive, the second component 172 can be omitted if necessary.

[0108] In some embodiments, a sealing member or layer is located between the port connector 160 and the second base 101 to ensure a fluid seal is formed between the orifice 174 and the port 103 or 104. In some embodiments, such as Figure 1CAs shown, an elastic member, such as a sealing ring 175, is located on the first member 170 or the second base 101. The elastic member 175 is compressed between the first member 170 and the second base 101 to form a seal. Alternatively, in some embodiments, an elastic sheet (similar to...) Figure 1A The layer 121 is located on the first member 170 or the second substrate 101 and is compressed between the first member 170 and the second substrate 101 to form a seal.

[0109] Sample preparation method

[0110] As described above, the enclosed chambers described herein can be formed on a substrate supporting a variety of samples. Examples of such samples include, but are not limited to, fresh tissues, such as biopsy sections, freshly frozen (FF) tissue sections, and formalin-fixed paraffin-embedded (FFPE) tissue sections. The methods described herein are typically used for one or more cycles of immunohistochemical (IHC) staining and imaging of biological samples enclosed in chambers formed as described above.

[0111] Figure 2 This is a flowchart illustrating a series of example steps for labeling and imaging FF tissue sections. In a first step 202, the FF tissue section is equilibrated to room temperature, for example by actively heating the tissue section or by passively allowing the tissue section to warm under ambient conditions. Next, in step 204, the tissue section is fixed by incubating it in an acetone bath at room temperature for a period of time, for example, 10 minutes. Then, in step 206, the tissue section is dried by allowing residual acetone in the tissue section to evaporate passively or by utilizing heating.

[0112] Next, in step 208, the tissue is hydrated in solution (e.g., in a solution such as potassium-buffered saline (PBS) for several minutes), and then in step 210, a tissue blocking step is performed. Next, in step 212, the tissue sections are incubated with one or more capture agents, each containing a primary antibody that specifically binds to a component (e.g., a target) in the sample. After incubation with the primary antibodies, the tissue sections are washed in step 214 and subjected to a first fixation in paraformaldehyde (PFA) solution in step 216.

[0113] Next, the tissue sections are cleaned in step 218 and undergo a second methanol fixation step 220. After cleaning again in step 222, the tissue sections undergo a bovine serum albumin (BSA) fixation step 224. Finally, the samples are cleaned again in step 226 and transported to storage in step 228, or subjected to one or more labeling and imaging cycles 230.

[0114] exist Figure 2Within the example steps shown, a closed chamber can be formed at any point in the workflow as described above after step 206. That is, at any time after residual acetone has been removed from the tissue section in step 206, a closed chamber can be formed, and subsequent sample processing steps can be performed by introducing fluids, reagents, and compositions (and their components) via one or more inlet ports 103 and removing fluids, reagents, and compositions (and their components) via one or more outlet ports 104.

[0115] Figure 3 This is a flowchart illustrating a series of example steps for labeling and imaging FFPE tissue sections. In the first step 302, the tissue sections are dewaxed using any of a variety of known dewaxing protocols (e.g., using consecutive xylene and ethanol washing).

[0116] Next, in step 304, antigen retrieval (AR) is performed. Many techniques exist for AR, and the selection of an appropriate technique depends on factors such as tissue type, fixation conditions, and the target epitope to be detected using IHC labeling. For example, common techniques that can be used for AR include heat-induced epitope retrieval (HIER) and proteolysis-induced epitope retrieval (PIER). These techniques can be performed on sample 151 on a first substrate 150 (e.g., on a microscope slide), which is standard practice. After AR, the tissue section is washed (e.g., in PBS solution) to remove residual AR reagent and is dried in step 306.

[0117] Subsequent steps 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, and 330 are similar to the corresponding steps 208-230 discussed above, so the discussion of these steps will not be repeated.

[0118] exist Figure 3 In the example steps shown, after step 302, a closed chamber can be formed at any point in the workflow as described above. That is, at any time after dewaxing, tissue sections can be rinsed in ethanol, acetone, toluene, or another non-aqueous solvent, residual solvent can be removed from the sample by drying, and a closed chamber can be formed. Subsequent sample processing steps can be performed by introducing fluids, reagents, and compositions through one or more input ports 103 and removing fluids, reagents, and compositions (and their components) through one or more output ports 104.

[0119] Sample Analysis Workflow

[0120] The above preparation steps can be used as corresponding to Figure 2 and 3Precursors to various labeling and imaging workflows in steps 230 and 330. It should be noted that in steps 228 and 328, the prepared samples may be transported to a storage facility within a closed chamber as described above. For example, the samples may be stored at a low temperature (e.g., about 4°C), retrieved from storage, and heated to room temperature for labeling and imaging at later dates and times (e.g., one or more hours, days, weeks, months, or even longer after preparation). In some embodiments, the prepared samples may undergo one or more labeling and imaging cycles before being sent to (or returned to) storage at the storage location or facility. The same samples still within the closed chamber may subsequently be retrieved for additional labeling and imaging cycles. Samples may be sent to and retrieved from storage multiple times, and some or all of the labeling and imaging steps may be repeated after sample storage intervals.

[0121] Furthermore, after the labeling and imaging cycle is completed (and in some embodiments, after the sample is retrieved from storage again), the chamber sealing the sample can be removed (e.g., by mechanically peeling off the second substrate 101), and the sample 151 can be further analyzed using additional analytical techniques. For example, genetic analysis can be performed on the sample 151, wherein the sample cells are lysed, DNA and / or RNA are collected, and the collected nucleic acids are sequenced, for example using any of a variety of different next-generation sequencing (NGS) technologies.

[0122] Compared to conventional IHC methods (where samples are typically not relabeled and imaged, especially after a period of storage), the ability to examine images obtained after one or more labeling and imaging cycles, and then repeat one or more labeling and imaging steps (e.g., to validate imaging parameters such as exposure time, and / or to detect weakly expressed sample components), offers significant advantages. Furthermore, the intermittent storage of samples, subsequent retrieval, and resumption of labeling and imaging cycles allow users to schedule analyses according to laboratory schedules and priorities.

[0123] Various different labeling and imaging workflows can be used in steps 230 and 330. Each workflow may include a single labeling and imaging step, or multiple labeling and imaging steps (e.g., multiple labeling and imaging cycles). Examples of these different types of workflows are described below.

[0124] Unordered IHC

[0125] In some embodiments, biological samples are labeled and imaged in a closed chamber in a single round. The label applied to the sample may be removed, or it may remain in the sample after imaging. To label the sample, a composition comprising one or more labels or stains (e.g., a fluid-based composition) is delivered into the closed chamber through one or more ports 103, and the label or stain interacts with the sample relatively rapidly after introduction, compared to conventional sample handling methods. The composition can be delivered via an automated fluid assembly or introduced manually by a technician.

[0126] In some embodiments, the composition delivered to the sealed chamber comprises a single type of marker or staining agent that binds to a specific type of component (e.g., a target) in the sample, such as a specific antigen or expressed protein marker. In other embodiments, the composition delivered to the sealed chamber comprises multiple different types of markers or staining agents, each specifically binding to a different type of component (e.g., a target) in the sample.

[0127] For example, in some embodiments, each different type of label may include a primary antibody or antibody fragment linked to a fluorescent labeler. The primary antibody or antibody fragment binds to a specific component (e.g., a target antigen or protein) in the sample. By exposing the labeler to incident light and obtaining a fluorescence emission image of the labeler, the spatial distribution and quantification of the corresponding component in the sample can be determined.

[0128] In some embodiments, tyrosine signal amplification (TSA) can be used to label samples for imaging. For a component of interest in the sample, a first composition comprising a primary antibody or antibody fragment specifically bound to that component is delivered to the sample. A second composition comprising a secondary antibody specifically bound to the primary antibody is then introduced. Linked to the secondary antibody is an enzyme catalyst (e.g., horseradish peroxidase (HRP)). Next, a third composition comprising tyramine labeled with a labeling agent is introduced, and the enzyme catalyst catalyzes the conversion of tyramine to tyramine radicals, which then bind to tyramine residues near the site of the primary antibody. In this way, a significant amplification of the fluorescence signal corresponding to the component can be achieved. Aspects of TSA are described, for example, in Toth et al., J. Histochem. Cytochem. 55(6); 545-554 (2007), the entire contents of which are incorporated herein by reference.

[0129] In some embodiments, instead of a direct conjugation between the secondary antibody and the enzyme catalyst, the secondary antibody may be biotinylated, and the enzyme catalyst (e.g., HRP) may be conjugated to streptavidin. The secondary antibody is first delivered to the sample, followed by the enzyme catalyst to selectively bind the catalyst to the secondary antibody, after which TSA is performed as described above.

[0130] In some embodiments, one or more staining agents may be applied to the sample. Staining may be performed according to a standard staining protocol before, during, or after one or more labeling and imaging cycles. The staining agent may be applied to the sample by delivering the staining solution into a sealed chamber, or alternatively, before the sealed chamber is formed. A variety of different staining agents may be used, including but not limited to DAPI, any Hoechst staining agent, hematoxylin and eosin, and combinations of multiple staining agents.

[0131] In all the aforementioned workflows, images of the label and staining agent in the sample are obtained using an imaging detector such as a CCD camera or a CMOS detector. Incident radiation from the source is directed to the stained / labeled sample, and light emitted from the sample is detected by the detector (e.g., imaging). In some embodiments, when the sample contains an absorptive (e.g., chromogenic) staining agent such as a counterstain, the light emitted from the sample typically corresponds to incident light transmitted from or reflected by the sample. When the sample contains a fluorescent staining agent (e.g., an immunofluorescent staining agent), the light emitted from the sample typically corresponds to fluorescence emitted by the staining agent in response to incident radiation.

[0132] When a sample contains a fluorescent label (e.g., a fluorescent portion that specifically binds to or is associated with a particular component (target) in the sample), incident radiation causes the label to emit fluorescence, which is imaged by a detector. The amount and spatial distribution of fluorescence emission can be used to quantitatively determine the relative amount of the component (target) at each location in the sample.

[0133] Examples of chromogenic staining agents that can be used include, but are not limited to, sulfonium-based dyes, such as fluorescein dyes and / or rhodamine dyes. Suitable examples of fluorescein and rhodamine dyes include, but are not limited to, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (generally known by the abbreviations FAM and F), 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE or J), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA or T), 6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G or G5), 6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110. Chromogenic staining agents may also include cyanide-based dyes. Suitable examples of such dyes include, but are not limited to, dyes Cy3, Cy5, and Cy7. The color-developing dyes can also be coumarin dyes (e.g., umbelliferone), benzoimide dyes (e.g., any Hoechst dye, such as Hoechst 33258), phenanthridine dyes (e.g., Texas Red), ethidium dyes, acridine dyes, carbazole dyes, benzoxazine dyes, porphyrin dyes, polymethystyl dyes (e.g., any BODIPY dye), and quinoline dyes.

[0134] Examples of immunofluorescence staining agents that can be used include, but are not limited to, pyrene, coumarin, diethylaminocoumarin, FAM, fluorescein chlorotriazine, fluorescein, Rl 10, JOE, R6G, tetramethylrhodamine, TAMRA, lysine, naphthalene fluorescein, Texas Red, Cy3, and Cy5.

[0135] Sequential staining multiple IHC

[0136] In addition to single-round simple or multiple IHC labeling, multiple IHC labeling and imaging can also be achieved through sequential labeling and imaging cycles. Figure 2 The workflow shown involves multiplex analysis with oligolabeled markers. In this type of analysis, a single incubation is performed with a primary antibody, followed by multiple rounds of oligolabeled markers via hybridization, marker imaging, dehybridization, and marker removal via a washing step.

[0137] The sample can be fixed and evaporated with acetone. Subsequently, a sealed chamber is formed from the sample within the chamber's internal volume. The primary incubation step can be performed by directly injecting a pipette into the sealed chamber. Fixation and label circulation, imaging, and label removal can be performed via an automated fluid system connected to the chamber's input and output ports 103 and 104, such as in Akoya. The system used in the platform (Akoya Biosciences, Menlo Park, CA).

[0138] After each round of marking, the sample can be imaged using a microscope with an automated stage. For example, a Nikon CFI S Plan Fluor ELWD 20XC with an NA of 0.45 can be used. Its focusing ring can be set to compensate for the thickness of the window and fluid layer between the sample and the objective lens. This compensation can be performed by adjusting before staining begins or by any preferred method, such that the objective lens is configured to properly image using the actual material and thickness present in the series of imaging steps. Other objectives can also be used, including but not limited to the Nikon CFI S Plan Fluor LWD20XC with an NA of 0.70.

[0139] Generally, the enclosed chamber described herein can be used in any workflow that allows for cyclic labeling, imaging, and label removal from a sample. Methods that include these steps after a single primary incubation are particularly advantageous because they reduce analysis time by introducing all primary antibodies into the sample in a single incubation step. However, methods involving multiple incubation steps employing primary antibodies (e.g., interleaved between labeling and imaging cycles) can also be implemented, as can methods that do not involve separate primary antibody incubation before imaging followed by additional labeling.

[0140] In some embodiments, one or more labeling and imaging cycles can be performed after primary antibody incubation, wherein label amplification occurs when the sample is labeled. A variety of different methods can be used for amplification, including oligo-mediated labeling combined with rolling circle amplification (RCA), branched or ligated amplification (BLA), oligo-ligated enzymatic amplification such as tyramine signal amplification (TSA) using oligoconjugated HRP, and combinations of these techniques.

[0141] One or more labeling and imaging cycles can also be performed where no label amplification occurs. Furthermore, in some embodiments, a first cycle of labeling, imaging, and label removal is performed using one type of label, such as a non-amplified oligolabel, followed by another cycle (immediately thereafter, or after an additional cycle) based on an amplified label, such as RCA, BLA, or TSA. In other embodiments, the cycle order is reversed.

[0142] In some embodiments that perform one or more labeling and imaging cycles, label removal can be omitted in any cycle, such as the final cycle. This saves time and effort and leaves the sample in a state where it may be imaged again in the future, with the last applied label present. It should be noted that, compared to some other workflows described herein, the TSA reaction deposits compounds in the sample that are not easily removed, and these compounds can interfere with subsequent labeling and / or imaging. Therefore, in some embodiments, it may be advantageous to use a TSA-based labeling reaction after other cycles have been completed.

[0143] In some embodiments, one or more samples (each enclosed in a corresponding chamber) are processed by a system having an automated labeling station with fluid components and an automated imaging station with an image detector. The system may optionally perform primary incubation and sample fixation steps, or these steps may be performed manually by a technician. The system performs one or more imaging cycles (e.g., labeling, imaging, and label removal) in a fully automated manner without user intervention. For the analysis of multiple samples, fluid and imaging operations can be interleaved between samples to improve efficiency. For example, when analyzing four samples, the first sample may be automatically incubated with a primary antibody, the system may deliver a probe as part of a labeling and imaging cycle to the second sample, the system may acquire an image of one or more labeling agents introduced into the third sample as part of a labeling and imaging cycle, and the system may deliver a label removal agent as part of a labeling and imaging cycle to the fourth sample.

[0144] IHC and histological staining

[0145] In some embodiments, one or more IHC cycles (e.g., labeling and imaging cycles) may be performed before histological staining of the sample. Alternatively, histological staining may be performed before one or more IHC cycles (e.g., labeling and imaging cycles) are performed on the sample.

[0146] A variety of histological dyes are available for this type of workflow. For example, by combining histological staining with one or more labeling and imaging cycles, a sample can be imaged with hematoxylin and eosin (H&E) staining, and can also be imaged with one or more IHC probes applied to the same sample and imaged sequentially. This allows pathologists to examine samples on the same sample with component quantification (i.e., biomarker data), select regions for analysis, and typically visualize structures.

[0147] Stains localized within selected tissue regions or materials can be used in image processing; for example, membrane-localized stains can facilitate the automated analysis of IHC images of the same sample. For instance, images corresponding to histological stains can be used as input data for cell segmentation algorithms.

[0148] Workflow efficiency

[0149] The enclosed chamber described herein allows slide-supported samples to be repeatedly stained and imaged. Furthermore, samples within the enclosed chamber can be easily handled without posing a risk to the sample and are readily connectable to manual or automated fluid components and devices.

[0150] Substrate with a closed chamber can be placed in a carrier, allowing for convenient and efficient processing of large numbers of samples. For example, a carrier with four or more such substrates can be connected to an automated fluid apparatus using a linked fluid connection. When labeling is complete, the sample can be disconnected and manually or automatically loaded into an automated imaging instrument. When scanning is complete, the batch of substrates can be reconnected to the fluid apparatus, the label is removed, and the label for the next cycle is introduced. This cycle can be repeated to perform multiplex IHC analysis with sequential labeling and imaging cycles.

[0151] Such a workflow offers several advantages. First, multiple samples can be processed in each step, whether manually or automatically. Second, while a given carrier with one set of samples is connected to an automated fluid assembly, an automated imaging station or device can scan another carrier with a different set of samples. Neither the imaging nor the fluid assembly is idle; the other is active and thus receives maximum utilization.

[0152] Furthermore, the sample is not connected to the fluid elements during imaging. This reduces or eliminates the risk of leakage or spillage from the imaging components. Additionally, the method described herein can be performed using various imaging stations (e.g., microscopes) because the imaging station does not need to be tightly integrated with the fluid components into a closed system. The second substrate and fluid in the microfluidic chamber covering the sample have optical effects and objectives, which have a “focusing ring” to compensate for varying amounts of glass or fluid in the optical path and can be used to acquire images of the sample.

[0153] In analyses involving one or more labeling and imaging cycles, the same sample can be imaged by multiple different imaging stations or systems within a given imaging cycle or in different imaging cycles. For example, images of one or more samples can be obtained using specialized microscopy techniques, such as confocal or multiphoton or super-resolution techniques, to obtain high-resolution images (and high-resolution quantitative information about biomarker expression) at locations where important sample components (targets) are present. In other cycles, the sample can be imaged at lower resolution and / or with different imaging stations or systems, such as whole-slide scanners.

[0154] Guide markers and imaging

[0155] The methods described herein can be used to implement guided labeling and imaging workflows. For example, after a closed chamber is formed around the sample, histological staining can be applied to the sample, or alternatively, a first labeling and imaging cycle can be performed with one or more different types of probes. Based on the properties of the sample (which can be determined at least in part from one or more images of histological staining and / or one or more labeling agents corresponding to the probes in the sample), a technician or automated controller performing the analysis software determines whether further analysis of the sample is necessary. In this way, the suitability of each sample can be confirmed at least in part based on its histological and / or IHC response before incurring further analytical costs and time. Degraded or unusable samples can then be identified.

[0156] In some embodiments, a technician or automated controller uses the sample information obtained in the foregoing manner to select one of a variety of possible staining / labeling and imaging protocols for further sample analysis. Based on the initially obtained information, samples from patients or other subjects can be guided along one of several staining / labeling and imaging procedures.

[0157] Alternatively, the sample can be processed through one or more IHC labeling cycles (e.g., labeling and imaging cycles). Unexpected or unpredictable findings obtained in this manner may suggest that further IHC measurements with one or more other labels would be valuable for a more comprehensive understanding of the sample. Additional steps, including any of those described herein, such as incubation with one or more primary antibodies, labeling, and imaging, can be performed to obtain these measurements.

[0158] After one or more cycles of IHC labeling and imaging, the information obtained from the sample images can be used to evaluate the quality of the acquired labeling and imaging. If defects are found, the sample can be reprocessed through one or more labeling and imaging cycles. This evaluation can be performed after each labeling and imaging cycle or at the end of all such cycles, and the sample images can be evaluated by a technician or automatically.

[0159] Oligonucleotide-based sample labeling

[0160] In some embodiments, oligonucleotide-based reagents are used to specifically label and image sample components. An example of such a reagent and its associated labeling workflow is available from Akoya Biosciences (Menlo Park, CA). Reagents. Certain aspects of oligonucleotide-based labeling reagents are described in, for example, U.S. Patent 10,370,698, the entire contents of which are incorporated herein by reference.

[0161] Oligonucleotide-based reagents can be used to perform multiple imaging cycles. Each imaging cycle may include the following steps: binding one or more probes to a biological sample, obtaining one or more images of the bound probes in the sample, and removing at least a portion of the bound probes from the sample before performing an additional cycle. It should be understood that while oligonucleotide-based reagents are well-suited for performing multiple imaging cycles, other types of reagents, including many others described herein, can also be used to perform such cycles.

[0162] To selectively label one or more components (targets) in a biological sample, the sample can first be incubated with one or more different types of capture agents. Each capture agent includes a binder that selectively binds to the sample component (target), and an oligonucleotide linked to the binder. Typical sample components include antigens and other protein markers, and the specific binders for these markers include, but are not limited to, antibodies and antibody fragments.

[0163] More generally, to target specific antigens, peptides, proteins, or other amino acid-containing species, the binder may include antibodies or antibody fragments. Antibodies or antibody fragments may include any different type of antibody, including but not limited to immunoglobulin G (IgG), immunoglobulin M (IgM), polyclonal antibodies, monoclonal antibodies, single-chain fragment variable (scFv) antibodies, nanobodies, antigen-binding fragments (Fab), and biantibodies. Antibodies and antibody fragments may be derived from mice, rats, rabbits, humans, camels, or goats. In some embodiments, antibodies or antibody fragments may be fed against antigens from humans, mice, rats, cattle, pigs, sheep, monkeys, rabbits, fruit flies, frogs, nematodes, or marmots. In some embodiments, antibodies or antibody fragments may be fed against antigens from animals, plants, bacteria, fungi, or protozoa.

[0164] The oligonucleotide linked to the binder is unique for each binder. Therefore, for example, in a composition containing three different types of trapping agents—each selectively binding to a different sample component—each type of trapping agent will have the same binder linked to the same oligonucleotide. The three types of binders will all be different, and the oligonucleotide of each type of trapping agent will differ from the others.

[0165] In some embodiments, the composition delivered to the biological sample includes only one type of trapping agent. However, in other embodiments, the composition includes multiple types of trapping agents. In particular, incubating samples with trapping agents is relatively time-consuming in typical sample analysis workflows. Therefore, by including multiple types of trapping agents (corresponding to multiple different targets or components in the sample) in the composition delivered to the sample, many (or even all) incubations can be performed simultaneously, significantly improving analytical efficiency.

[0166] In some embodiments, the composition delivered to the sample may include two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, ten or more, fifteen or more, twenty or more, twenty-five or more, thirty or more, thirty-five or more, forty or more, fifty or more, sixty or more, seventy or more, eighty or more, or even more) different types of trapping agents.

[0167] Each oligonucleotide linked to a trapping agent comprises a sufficient number of nucleotides such that the probe (discussed below) selectively binds only to one type of trapping agent. In some embodiments, the oligonucleotide comprises 15 or more (e.g., 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 55 or more, or even more) nucleic acids.

[0168] After one or more capture agents bind to the sample, various types of capture agents are labeled and imaged in one or more labeling, imaging, and label removal cycles. Each such cycle comprises three steps. In the first step, the sample is exposed to a composition containing one or more probes (e.g., by introducing the composition into a chamber formed as described above that encloses the sample). Each composition may include one type of probe, or more than one type of probe. Each type of probe selectively binds to only one type of capture agent in the sample.

[0169] By including multiple types of probes in the composition, multiplex sample labeling and imaging can be performed. Each different type of probe in the composition binds simultaneously to a different type of trapping agent in the sample, significantly reducing the time consumed in sample-probe incubation. In some embodiments, the composition may include one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, ten or more, or even more) different types of probes.

[0170] Each type of probe consists of an oligonucleotide linked to a label. The oligonucleotide and label are identical for single-probe types. However, the oligonucleotide and label differ between different probe types. Because each type of probe selectively binds to only one type of trap, a specific label is associated with a specific trap (and therefore with a specific sample component or target). In this way, each type of label acts as a reporter for different sample components.

[0171] Each probe oligonucleotide contains a sufficient number of nucleic acids to achieve specific binding between the trap and the probe pair. Probe oligonucleotides typically contain 15 or more (e.g., 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, or even more) nucleic acids.

[0172] In order to specifically bind to a particular type of capture agent, the oligonucleotide of each type of probe is at least partially complementary to the oligonucleotide of the corresponding capture agent. As used herein, “at least partially complementary” means that there are no more than five perfectly complementary mismatches between the nucleic acid sequence of the probe oligonucleotide and at least a portion of the capture agent oligonucleotide.

[0173] A variety of different labeling agents can be linked to probe oligonucleotides. In some embodiments, for example, the labeling agent includes a fluorescent portion having a different emission center wavelength or emission bandwidth, or both.

[0174] The probe can bind to the trapping agent via hybridization between corresponding oligonucleotides. Hybridization is then easily reversed, allowing removal of the probe from the sample. Alternatively, other methods for associating the probe and the trapping agent can be used. In some embodiments, for example, the probe can bind to a specific trapping agent via a ligation or primer extension reaction.

[0175] In the next step of the cycle, the probes bound to the trapping agent in the sample are imaged. Specifically, a fluorescence image corresponding to the emission from each bound probe is obtained. In some embodiments, a fluorescence image including contributions from essentially only one type of probe can be obtained, for example, by emission filtering. In some embodiments, the fluorescence image includes significant contributions from multiple probes in the sample, and the individual contributions can be separated using techniques such as spectral unmixing.

[0176] In the third step of the cycle, the probe is at least partially removed from the sample. In some embodiments, the removal of one or more probes is completed. In some embodiments, a residual amount of one or more probes remains in the sample. For the terminal cycle (i.e., the last cycle in the analysis workflow), the probe may remain in the sample to facilitate subsequent re-imaging of the sample, for example, after a period of storage.

[0177] Typically, removing a probe from a sample involves dehybridizing the probe's oligonucleotides and a corresponding trapping agent. Various techniques can be used for dehybridization. In some embodiments, dehybridization can be performed, for example, by heating the sample to denature the hybridized oligonucleotides. In some embodiments, the sample may be exposed to one or more dehybridization agents. Examples of such agents include, but are not limited to, liquid-dissociating agents such as guanidine hydrochloride, guanidine thiocyanate, and urea.

[0178] After the probe is removed (or partially removed), an additional cycle of labeling, imaging, and label removal can be performed to obtain quantitative information about additional components (e.g., biomarkers) in the sample.

[0179] Analysis of sample components using oligonucleotide-based reagents can also be combined with TSA-based signal amplification. For example, methods and reagents for TSA amplification using oligonucleotide-based reagents are described in PCT patent application No. PCT / US2020 / 016667, filed February 4, 2020, the entire contents of which are incorporated herein by reference.

[0180] Automated systems for the analysis of single and multiple samples

[0181] The methods described herein can be implemented in a variety of different analytical systems, including those that perform some or all of the steps in a semi-automatic or fully automatic manner. An example of such a system is... Figure 5The diagram is schematically shown. System 500 includes a storage unit 502, a marker station 504, an imaging station 506, and a translation device 510. Each of these components is connected to a controller 508, which includes one or more electronic processors that perform control functions associated with the controller 508, and may also perform any other analytical functions described herein.

[0182] Translation device 510 includes a slide processor 512 attached to a single slide containing a sample within a sealed chamber for transporting the slide between different locations within the system. An example of a slide with a sealed chamber and a sample within it is shown in... Figure 5 The substrate is designated as substrate 550. The substrate processor 512 can be implemented in various ways. In some embodiments, for example, the substrate processor 512 is a gripper and includes one or more arms or fingers that apply pressure to the surface of the substrate 550 to lift and transport the substrate 550. In some embodiments, the substrate processor 512 includes a member having one or more suction ports that uses decompression to lift a single substrate 550. In some embodiments, the substrate processor 512 includes one or more members inserted beneath the substrate 550 to lift the substrate. Typically, the substrate processor 512 can allow rotational displacement of the substrate 550 about three orthogonal axes, as well as translational displacement along the three orthogonal axes.

[0183] The translation device 510 may also include a track or conveyor 513 that transports individual slides 550 or slide containers between locations of the system 500. In some embodiments, the track 513 is a linear track that moves back and forth in a single direction between locations. In some embodiments, the track 513 is a continuous track (e.g., circular, elliptical, or other continuous shape) that circulates between locations in the system.

[0184] As described above, during operation, controller 508 can send appropriate control signals to translation device 510 to retrieve one or more slides 550 from storage unit 502 and deposit one or more slides into storage unit 502. Furthermore, controller 508 can send control signals to translation device 510 to activate labeling station to deliver fluids, reagents, and compositions to and from the chambers of slide 550. Labeling station 504 includes fluid device 514 connected to one or more reservoirs 518 and one or more pumps and / or vacuum sources 519. Fluid device 514 includes one or more fluid conduits 116 (e.g., syringes, tubes) selectively coupled to ports 103 and / or 104 of the chambers of slide 550. During system operation, controller 508 signals translation device 510 to position slide 550 within labeling station 504 and signals fluid device 514 to connect one or more fluid conduits to one or more ports 103 and / or 104. In this manner, fluids, reagents, and compositions are delivered from reservoir 518 into the chamber of slide 550, and fluids, reagents, and compositions (and their components) are removed from the chamber, enabling controller 508 to automate any staining and labeling operations described herein. Under the control of controller 508, labeling station 504 can perform any sample preparation steps described herein.

[0185] Imaging station 506 includes a radiation source 520, an objective lens 524, a beam splitter 522, and an image detector 526. The radiation source 520 may include any one or more of a variety of different sources, including but not limited to LEDs, laser diodes, metal halide sources, incandescent sources, and fluorescent sources. The image detector 526 may include one or more different detector types, including but not limited to CCD detectors and CMOS detectors. During operation of system 500, in order to obtain an image of the sample within the chamber of slide 550, controller 508 activates translation device 510 to position slide 550 within imaging station 506. Controller 508 then sends a control signal to components of the imaging station to activate source 520 to deliver illumination radiation to the sample, which passes through beam splitter 522 and objective lens 524 and is incident on the sample. The emitted light from the sample passes through objective lens 524, is reflected from beam splitter 522, and is incident on image detector 526, which measures the image of the emitted light.

[0186] exist Figure 5In this embodiment, the imaging station is configured to acquire fluorescent or reflected light images of the sample. However, it should be understood that in some embodiments, detector 526 may be located on the side of slide 550 opposite to source 520 to measure transmitted light images of the sample. In some embodiments, imaging station 506 includes multiple detectors for measuring transmitted and reflected light or emitted light (e.g., fluorescence) images of the sample. Under the control of controller 508, imaging station 506 can acquire images of any type corresponding to any type of staining agent, probe, and labeling agent described herein.

[0187] As previously described, each step in sample analysis consumes a certain amount of time, and improved efficiency can be achieved by translating multiple slides 550 between multiple locations within system 500 and performing multiple operations. For example, during the analysis of multiple slides 550, a first slide containing a first sample in a chamber can be positioned at a labeling station, and a fluid device can deliver one or more probes to the sample and incubate the sample with the delivered probes. Simultaneously, a second slide containing a second sample in a chamber can be positioned at an imaging station to obtain one or more images of the staining or labeling agent in the sample. A third slide containing a third sample in a chamber can be positioned at a labeling station, where a controller 508 activates one or more pumps and / or a vacuum source to remove fluid, reagents, and / or compositions (and their components) from the chamber of the third slide. A fourth slide containing a fourth sample in a chamber can be positioned at a labeling station, where a controller 508 activates a fluid device 514 to deliver a composition containing one or more primary antibodies into the chamber and incubate the fourth sample with the antibody composition. Other slides 550 can also be processed simultaneously—performing any of the operations discussed herein.

[0188] As each slide reaches the end of one or more steps in the preparation or analysis workflow, it is transported by translation device 510 to different locations within the system, such as different stations or different locations within the same station. In this way, system 500 can analyze multiple samples simultaneously, ensuring that the duty cycle of system 500 components remains relatively high, increasing the system's total throughput for multiple samples compared to the simple linear processing of a single sample.

[0189] Figure 6 An example of controller 508 is shown, which can be used with the systems and methods disclosed herein. Controller 508 may include one or more processors 402, memory 404, storage device 406, and interface 408 for interconnection. Processor 402 may process instructions that execute within the controller, including instructions stored on memory 404 or storage device 406. For example, instructions may instruct processor 402 to perform any of the analysis and control steps disclosed herein.

[0190] Memory 404 may store executable instructions from processor 402, information about system parameters such as excitation and detection wavelengths, and measured spectral image information. Storage device 406 may be a computer-readable medium, such as a floppy disk device, hard disk device, optical disk device, magnetic tape device, flash memory or other similar solid-state storage device, or a device array, including devices in a storage area network or other configuration. Storage device 406 may store instructions executable by the processor 402, as well as any other information that may be stored in memory 404.

[0191] In some embodiments, controller 508 may include a graphics processing unit to display graphical information (e.g., using a GUI or text interface) on an external input / output device such as display 416. The graphical information may be displayed by a display device (e.g., a CRT (cathode ray tube) or LCD (liquid crystal display) monitor) to display any information, such as the spectra and images disclosed herein for measurement and calculation. Users may provide input to controller 508 using input devices such as keyboards, pointing devices, touchscreens, or voice recognition devices.

[0192] Users of system 500 can provide various types of instructions and information to controller 508 via input devices. For example, instructions and information may include information on any parameters (e.g., staining agents, labels, probes, reagents, conditions) related to any staining and labeling protocols described herein, calibration information for quantitative analysis of sample images, and instructions following manual analysis of sample images by a technician. Controller 508 can use any of these various types of information to perform the methods and functions described herein. It should also be noted that any of these types of information can be stored (e.g., in storage device 406) and recalled by controller 508 when needed.

[0193] The methods disclosed herein can be implemented by controller 508 by executing instructions in one or more computer programs executable and / or interpretable by controller 508. These computer programs (also referred to as programs, software, software applications, or code) include machine instructions for a programmable processor and can be implemented using high-level procedural and / or object-oriented programming languages ​​and / or assembly / machine languages. For example, a computer program may contain instructions that can be stored in memory 404, storage unit 406, and / or on a tangible computer-readable medium, and executed by processor 402 as described herein. As used herein, the term "computer-readable medium" refers to any computer program product, apparatus, and / or device (e.g., disk, optical disk, memory, programmable logic device (PLD), ASIC, and electronic circuit) used to provide machine instructions and / or data to a programmable processor, including machine-readable media that receive machine instructions.

[0194] Example

[0195] Example: Multiple IHC labeling and histochemical staining

[0196] To demonstrate the effectiveness of the method described herein, experiments were conducted in which FFPE tissue samples were analyzed by multiplex IHC labeling and histochemical staining. Samples mounted on microscope slides were heated at 55°C for 30 minutes. The samples were then equilibrated at room temperature for approximately 10 minutes and dewaxed. Next, antigen recovery was performed with tri-ethylenediaminetetraacetic acid (tri-EDTA), followed by cooling for 10 minutes. The samples were then washed three times with deionized water and incubated in acetone for 10 minutes. After being removed from the acetone bath and dried to remove residual acetone, the samples were then used… Figure 1B gaskets, windows and such Figure 1C The two-port connector shown forms a closed chamber on the microscope slide.

[0197] The samples were then incubated twice for two minutes each in a buffer solution containing EDTA, DPBS, BSA, and NaN3 (“S1”). Afterward, the samples were incubated for 30 minutes in a buffer solution containing EDTA, DPBS, BSA, NaN3, NaCl, and NaH2PO3 (“S2”). Finally, the samples were incubated for 3 hours in a primary antibody composition comprising buffer S2, an IgG rat-derived N-blocker, an IgG mouse-derived J-blocker, cleaved salmon DNA as an S-blocker, an oligonucleotide-based G-blocker, and a 2 μg / mL primary antibody mixture comprising three different primary antibodies, each linked to three different oligonucleotides.

[0198] After incubation with the primary antibody, the samples were washed twice in S2 buffer and fixed in a solution of S2 buffer and 1.6% PFA for approximately 4 minutes. Next, the samples were washed three times in DPBS and fixed in methanol for approximately 5 minutes. Then, the samples were washed three times in DPBS and fixed in a solution of DPBS and BS3 for approximately 6 minutes, followed by three more washes in DPBS.

[0199] Finally, the samples were stained and imaged sequentially with DAPI, FITC, and three IHC probes, each containing an oligonucleotide that selectively hybridizes to a primary antibody oligonucleotide and contains a different fluorescent labeling agent.

[0200] Figures 4A-4E Images of each stain and label applied to the sample are shown. Figure 4A It is an image showing the distribution of DAPI within the sample. Figure 4B It is an image showing the FITC distribution within the sample. Figure 4C-4ECD68 (corresponding fluorescent label Cy5) and whole-cell keratin (corresponding fluorescent label) are shown. Distribution of E-cadherin (750) and E-cadherin (corresponding fluorescent marker Atto 550). Each of these markers is clearly visible in the corresponding image.

[0201] Other embodiments

[0202] While this disclosure describes specific implementations, these should not be construed as limiting the scope of the disclosure, but rather as descriptions of features in certain embodiments. Features described in the context of a single embodiment may also generally be implemented in combination in a single embodiment. Conversely, various features described in the context of a single embodiment may also be implemented individually or in any suitable sub-combination in multiple embodiments. Furthermore, although features may be described above as existing in certain combinations, or even initially claimed in this way, one or more features in a claimed combination may generally be removed from that combination, and a claimed combination may refer to a sub-combination or a variation of a sub-combination.

[0203] In addition to the embodiments explicitly disclosed herein, it should be understood that various modifications may be made to the described embodiments without departing from the spirit and scope of this disclosure. Therefore, other embodiments are within the scope of the following claims.

Claims

1. A method for analyzing biological samples, the method comprising: Obtain biological samples mounted on the first substrate; A second substrate is fixed to a first substrate to form a closed chamber on the first substrate, and a biological sample is located within the internal volume of the closed chamber. The second substrate includes a plurality of inlet ports, each inlet port extending through the second substrate and positioned such that the impedance of fluid flow along the fluid flow path corresponding to each inlet port is substantially the same. as well as Execute multiple imaging cycles, where each imaging cycle includes: (a) Binding probes to biological samples; (b) Obtaining images of the probes bound in the biological sample; and (c) Remove at least a portion of the probe from the biological sample. The thickness of the internal volume between the first and second substrates is 250 micrometers or less.

2. The method according to claim 1, wherein, The first substrate is formed of a substantially transparent material.

3. The method according to claim 1, wherein, The first substrate is formed of glass or plastic material.

4. The method according to claim 1, wherein, The first substrate includes a microscope slide.

5. The method according to claim 1, wherein, The second substrate includes a window.

6. The method of claim 1, further comprising positioning a gasket between the first and second substrates.

7. The method according to claim 6, wherein, The gasket is formed of an adhesive material.

8. The method of claim 1, further comprising, before fixing the second substrate to the first substrate: Incubate biological samples in acetone; Removal of residual acetone from biological samples; and After removing the residual acetone, the second substrate is fixed to the first substrate.

9. The method of claim 8, further comprising, prior to incubating the biological sample in acetone: Removal of paraffin from biological samples; and Expose biological samples to antigen retrieval agents.

10. The method according to claim 8, wherein, The sample is a fresh frozen sample.

11. The method according to claim 9, wherein, The sample was a formalin-fixed paraffin-embedded sample.

12. The method of claim 11, further comprising applying a staining agent to the biological sample prior to performing the plurality of imaging cycles.

13. The method according to claim 12, wherein, The staining agent includes at least one selected from the group consisting of counterstaining agents, chromogenic staining agents, and immunofluorescence staining agents.

14. The method according to claim 12, wherein, The staining agent includes at least one of DAPI and Hoechst staining agent.

15. The method according to claim 1, wherein, The second substrate includes a first port formed by a first hole extending through the thickness of the second substrate and a second port formed by a second hole extending through the thickness of the second substrate, wherein the composition is introduced into the internal volume through the first port.

16. The method according to claim 15, wherein, The minimum distance between the first and second ports, measured in the plane of the second substrate, is 20 mm or greater.

17. The method according to claim 15, wherein, During each imaging cycle, the probe is bound to a biological sample by introducing a composition containing the probe through the first port and removing a component of the composition through the second port.

18. The method of claim 15, further comprising, prior to performing the plurality of imaging cycles, binding the trapping agent to the biological sample by introducing a composition comprising the trapping agent through the first port.

19. The method according to claim 18, wherein, The trapping agent includes a binder that selectively binds to sample components and an oligonucleotide linked to the binder.

20. The method according to claim 19, wherein, The binding agent includes an antibody or an antibody fragment.

21. The method according to claim 19, wherein, The oligonucleotide contains at least 15 nucleic acids.

22. The method according to claim 19, wherein, The composition comprises multiple different types of trapping agents, each type of trapping agent comprising a different type of binder that selectively binds to different sample components, and oligonucleotides specific to each type of binder.

23. The method according to claim 22, wherein, The composition contains at least five different types of trapping agents.

24. The method according to claim 22, wherein, The composition contains at least 20 different types of trapping agents.

25. The method according to claim 22, wherein, Each different type of trapping agent is incorporated into the biological sample by simultaneously introducing a composition containing each different type of trapping agent into the internal volume of the enclosed chamber.

26. The method according to claim 19, wherein, The probe comprises an oligonucleotide that is at least partially complementary to the oligonucleotide of the trapping agent, and a labeling agent linked to the oligonucleotide.

27. The method according to claim 26, wherein, The probe contains at least 20 nucleic acids in its oligonucleotide.

28. The method according to claim 26, wherein, The labeling agent includes a fluorescent portion.

29. The method according to claim 22, wherein, Binding the probe to a biological sample includes binding one or more different types of probes to the biological sample, wherein each different type of probe includes a different type of oligonucleotide linked to a labeler specific to each different type of oligonucleotide.

30. The method according to claim 29, wherein, The one or more different types of probes include at least three different types of probes.

31. The method according to claim 29, wherein, One or more different types of probes are simultaneously bound to a biological sample by introducing a composition containing one or more different types of probes into the internal volume of the sealed chamber.

32. The method according to claim 29, wherein, One or more different types of probes bind to corresponding different types of capture agents in biological samples.

33. The method according to claim 32, wherein, The one or more different types of probes hybridize with corresponding different types of capture agents in the biological sample.

34. The method according to claim 26, wherein, The oligonucleotide of the probe hybridizes with the oligonucleotide of the trapping agent, and wherein removing at least a portion of the probe from the biological sample includes dehybridizing at least a portion of the probe from the trapping agent.

35. The method of claim 34, further comprising dehybridizing the at least a portion of the probe from the trapping agent by heating a biological sample.

36. The method of claim 34, further comprising dehybridizing the at least a portion of the probe from the trapping agent by heating the biological sample by introducing at least one dehybridizing agent into the internal volume of the sealed chamber.

37. The method according to claim 36, wherein, The at least one dehybridizing agent includes a liquid-dissolving agent.

38. The method according to claim 1, wherein, The thickness of the internal volume is 100 micrometers or less.

39. The method according to claim 1, wherein, The internal volume is 0.20 cm³. 3 Or smaller.

40. The method according to claim 1, wherein, The internal volume is 0.10 cm³. 3 Or smaller.

41. The method of claim 15, further comprising connecting a port connector to at least one of the first and second ports before introducing the composition into the internal volume of the enclosed chamber.

42. The method according to claim 41, wherein, The port connector includes a first component that includes at least one fluid passage aligned with one of the first and second ports.

43. The method according to claim 42, wherein, Connecting the port connector to at least one of the first and second ports includes adhering the port connector to the surface of the second substrate.

44. The method according to claim 42, wherein, The port connector includes a second component, and wherein connecting the port connector to at least one of the first and second ports includes first and second components for connecting the port connector to secure the port connector in place relative to the second base.

45. The method according to claim 42, wherein, At least one of the first and second components includes at least one magnet, and wherein the first and second components are positioned such that at least one magnet holds the first and second components in place relative to the second substrate.

Images