Multiplexed tissue imaging

Abstract

To provide a system and a method for imaging a biological sample, capable of comparatively reducing the number of probes and scavengers used in detecting a sample component by enclosing a sample in a comparatively small volume of chamber and keeping labeling and the period of an imaging cycle at a manageable level.SOLUTION: An apparatus attaches 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. Flow of reagents within the chamber can be laminar and highly uniform across the sample surface to achieve staining that is free of gradients. The sample can be imaged prior to, during, or after any given processing step. Methods for staining and imaging a sample can be implemented through one or more rounds of labeling, label removal or erasure and imaging. Samples can be imaged many times to achieve very high multiplexing levels. Samples can be placed on an imaging apparatus at various times and removed from the imaging apparatus for certain steps.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 861,991, filed on Jun. 14, 2019, the entire content of which is incorporated herein by reference.

[0002] This disclosure relates to systems and methods for imaging biological samples.

Background Art

[0003] Immunohistochemistry (IHC) is used to detect a target molecular in cells or tissues, using an antibody that binds to a specific target of interest and a labeling system that enables the detection of the antibody. Various optical labeling systems have been devised, such as colored dyes or precipitates, fluorescent molecules, and various nanoparticles. IHC is very valuable in the research, diagnosis, and treatment guidance of various diseases.

[0004] To understand a particular biological or medical process, it may be important to know the amount or presence of several molecular species within each cell. This is because the role or function of a particular cell population is not easily determined by imaging a single molecular species.

[0005] Multiplex IHC techniques have been developed that can detect or quantify multiple molecular targets, including targets that are localized within the same compartment of a particular cell.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

[0007] [Non-Patent Document 1] Glass et al., J. Histochem Cytochem. 57(10):899 - 905(2009) [Non-Patent Document 2] eLife 2018, 7:e31657 by Lin et al. [Non-Patent Document 3] Toth et al., J. Histochem. Cytochem. 55(6);545 - 554(2007) [Summary of the Invention] [Means for Solving the Problems]

[0008] The present disclosure features methods and systems for multiplex imaging of biological samples. The method can include, for example, forming a sealed chamber around a sample on a substrate such as a microscope slide. The chamber volume is relatively small, and reagents can be introduced into the chamber to perform multiple labeling and imaging cycles on the sample. During each cycle, the sample can be labeled with one or more probes, and one or more images of the probes can be acquired. Thereafter, the probes can be removed and additional probes can be conjugated to the sample and the images. Each type of probe typically selectively binds to one type of component (e.g., one type of protein marker) within the sample, and the corresponding image of that type of probe provides quantitative information regarding the distribution and amount of the components in the sample. By repeating the labeling and imaging cycles, a relatively large number (e.g., 20 or more, 40 or more, or more) of different sample components can be imaged and quantified.

[0009] Enclosing the sample in a relatively small volume chamber can reduce the amount of probes and capture agents used for detecting sample components, and the duration of the labeling and imaging cycles can be maintained at a manageable level, which is an important consideration for highly multiplexed analysis. Further, due to the relatively small chamber volume, the composition of the probes and capture agents can flow in a laminar flow over the entire biological sample, ensuring that non-uniform staining (e.g., due to vortices, gradients, and other non-laminar flow phenomena) is controlled or even eliminated. Additionally, with a constant chamber volume and a very small dead volume (less than 5 microliters), an accurate amount of the composition of the probes and capture agents can be dispensed into the chamber, reducing waste and overflow. During sample processing, the fluid can be moved over the sample or back and forth within the sealed chamber to facilitate the interaction between the reagents in the fluid and the components (such as targets) near the sample surface.

[0010] Another important advantage results from the ability to disconnect the sealed chamber (and the sample therein) from various stations or locations within the processing system and even from the system itself for storage. Since the sample is enclosed within the chamber, the sample and the substrate supporting the chamber can be moved to different locations and within the tube location, and the sample can be stored within the chamber. Subsequently, the sample can be retrieved from the storage location days, weeks, months, or more later and subjected to further processing and / or imaging steps. Additionally, the components of the chamber may be removed, and the sample can be subjected to different types of analysis (e.g., DNA and / or RNA extraction and sequencing).

[0011] In one aspect, the present disclosure is a method for analyzing a biological sample, comprising the steps of obtaining a biological sample attached to a first substrate, attaching a second substrate to the first substrate to form a sealed chamber on the first substrate and disposing the biological sample within the internal volume of the sealed chamber, and performing a plurality of imaging cycles, each imaging cycle comprising (a) binding a probe to the biological sample, (b) obtaining an image of the bound probe in 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 substrate and the second substrate is 250 micrometers or less.

[0012] In another aspect, the present disclosure is a method for analyzing a biological sample, comprising the steps of: obtaining a biological sample attached to a first substrate; attaching a second substrate to the first substrate to form a sealed chamber on the first substrate and placing the biological sample within the internal volume of the sealed chamber; and performing a plurality of imaging cycles, each imaging cycle including: (a) binding a probe to the biological sample; (b) obtaining an image of the bound probe in the biological sample; and (c) removing at least a portion of the probe from the biological sample, wherein the first substrate and the second substrate are substantially transparent and the imaging window formed by the first substrate and the second substrate has a field of view of at least 500 mm 2 and the method is characterized by including the step of:

[0013] In another aspect, the present disclosure is a method for analyzing a biological sample, comprising the steps of: obtaining a biological sample attached to a first substrate; attaching a second substrate to the first substrate to form a sealed chamber on the first substrate and placing the biological sample within the internal volume of the sealed chamber; and performing a plurality of imaging cycles, each imaging cycle including: (a) binding a probe to the biological sample; (b) obtaining an image of the bound probe in 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 passing through the first substrate and connected to the internal volume, and a second port defining a second channel passing through the first substrate and connected to the internal volume, and the closest distance between the first port and the second port measured in the plane of the second substrate is 25 mm or more, and the method is characterized by including the step of:

[0014] In another aspect, the present disclosure is a method for analyzing a biological sample, comprising the steps of: obtaining a biological sample attached to a slide; contacting the slide with a gasket, wherein the gasket includes an opening and the sample is disposed within the opening; and contacting the gasket with a window, thereby enclosing the sample within an internal volume surrounded by the slide, the gasket, and the window, wherein the window defines an observation region having a length of at least 25 mm and a width of at least 15 mm.

[0015] In another aspect, the present disclosure features a system for analyzing a biological sample, 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. The controller is configured such that during operation of the system, the controller: (a) obtains a first biological sample enclosed within a first chamber formed by a first substrate, a second substrate, and a gasket disposed between the first and second substrates, the first substrate being disposed on a first substrate; and a second biological sample enclosed within a second chamber formed by a third substrate, a fourth substrate, and a gasket disposed between the third and fourth substrates, the third substrate being disposed on a third substrate; (b) uses the translation device to place the first biological sample at the labeling station and actuates the fluid device to deliver a first labeling agent to the first biological sample within the first chamber; (c) uses the translation device to place the first biological sample at the imaging station and actuates the image detector to obtain an image of the first labeling agent within the first biological sample; (d) places the first biological sample at the imaging station, uses the translation device to place the second biological sample at the labeling station, and actuates the fluid device to deliver a second labeling agent to the second biological sample within the second chamber; (e) removes the first biological sample from the imaging station; (f) places the second biological sample at the imaging station and actuates the image detector to obtain an image of the second labeling agent within the second biological sample; (g) places the first biological sample at the labeling station, actuates the fluid device to remove at least a portion of the first labeling agent from the first biological sample, and delivers a third labeling agent to the first biological sample.

[0016] Any embodiment of the methods and systems disclosed herein may include any one or more of the following features.

[0017] The first substrate may be formed of a substantially transparent material, such as a glass or plastic material. The first substrate may be a microscope slide. The second substrate may be a window.

[0018] The method may further include the step of disposing a gasket between the first substrate and the second substrate. The gasket may be formed of an adhesive.

[0019] The method may include the steps of incubating a biological sample in acetone, removing residual acetone from the biological sample, and attaching the second substrate to the first substrate after removing the residual acetone, before the step of attaching the second substrate to the first substrate. The method may include the steps of removing paraffin from the biological sample and exposing the biological sample to an antigen retrieval agent, before the step of incubating the biological sample in acetone.

[0020] The sample may be a fresh frozen sample. The sample may be a formalin-fixed paraffin-embedded sample.

[0021] The method may include the step of applying a dye to the biological sample before performing a plurality of imaging cycles. The dye may include at least one element selected from the group consisting of a contrast dye, a chromogenic dye, and an immunofluorescent dye. The dye may include at least one of DAPI and a Hoechst dye.

[0022] The second substrate may include a first port formed by a first opening extending through the thickness of the second substrate and a second port formed by a second opening extending through the thickness of the second substrate, and a composition is introduced into the internal volume through the first port. The minimum distance between the first port and the second port measured in the plane of the second substrate may be 20 mm or more.

[0023] During each imaging cycle, a composition containing a probe may be introduced through the first port and components of the composition may be removed through the second port, whereby the probe can be bound to the biological sample. The method may include the step of binding a capture agent to the biological sample by introducing a composition containing the capture agent through the first port before performing a plurality of imaging cycles.

[0024] 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.

[0025] The composition may include a plurality of different types of capture agents, and each of the different types of capture agents includes a different type of binder that selectively binds to a different sample component and an oligonucleotide specific to each different type of binder. The composition may include at least 5 different types of capture agents (e.g., at least 20 different types of capture agents). By introducing a composition that simultaneously includes each of the different types of capture agents into the internal volume of a sealed chamber, each of the different types of capture agents can be bound to a biological sample.

[0026] The probe may include an oligonucleotide that is at least partially complementary to the oligonucleotide of the capture agent and a labeling agent linked to the oligonucleotide. The oligonucleotide of the probe may include at least 20 nucleic acids.

[0027] The labeling agent may include a fluorescent moiety.

[0028] The step of binding the probe to a biological sample includes the step of binding one or more different types of probes to the biological sample, and each of the different types of probes may include a different type of oligonucleotide linked to a labeling agent specific to each different type of oligonucleotide.

[0029] One or more different types of probes can include at least three different types of probes. By introducing a composition comprising one or more different types of probes into the internal volume of a sealed chamber, the one or more different types of probes can be simultaneously bound to a biological sample. The one or more different types of probes can bind to corresponding different types of capture agents in the biological sample. The one or more different types of probes can hybridize to corresponding different types of capture agents in the biological sample.

[0030] The step of hybridizing the probe oligonucleotide to the capture agent oligonucleotide and removing at least a portion of the probe from the biological sample can include the step of dehybridizing at least a portion of the probe from the capture agent. The method can include the step of dehybridizing at least a portion of the probe from the capture agent by heating the biological sample and / or by introducing at least one dehybridizing agent into the internal volume of the sealed chamber. The at least one dehybridizing agent can include a chaotropic agent.

[0031] The thickness of the internal volume can be 100 micrometers or less. The internal volume can be 0.20 cm 3 or less (for example, 0.10 cm 3 or less).

[0032] The method may include connecting a port coupler to at least one of the first and second ports before introducing the composition into the internal volume of the sealed chamber. The port coupler may include a first member including at least one fluid channel aligned with one of the first port and the second port. The step of connecting the port coupler to at least one of the first port and the second port may include adhering the port coupler to the surface of the second substrate. The port coupler may include a second member, and the step of connecting the port coupler to at least one of the first and second ports may include connecting the first member and the second member of the port coupler to fix the port coupler in a predetermined position relative to the second substrate. At least one of the first member and the second member may include at least one magnet, and the first and second members may be arranged such that the at least one magnet fixes the first and second members in a predetermined position relative to the second substrate.

[0033] Embodiments of the method and system may also include any other features disclosed herein, and may include any combination of features, including combinations of features described separately in connection with different embodiments, unless otherwise specified.

[0034] Some embodiments described herein relate to a computer storage product having a non-transitory computer-readable medium (also referred to as a non-transitory processor-readable medium) having instructions or computer code thereon for performing various computer-implemented operations. The computer-readable medium (or processor-readable medium) is non-transitory in the sense that it does not include the transient propagation signal itself (e.g., a propagating electromagnetic wave carrying information on a transmission medium such as space or a cable). The medium and the computer code (also referred to as code) can be designed and constructed for a particular purpose. Examples of non-transitory computer-readable media include magnetic storage media such as hard disks, floppy disks, and magnetic tapes; optical disks such as compact discs / digital video discs (CD / DVDs), compact disc-read only memory (CD-ROMs), and holographic devices; magneto-optical storage media such as optical disks; carrier signal processing modules; hardware devices specially configured to store and execute program code, such as application specific integrated circuits (ASICs), programmable logic devices (PLDs), read only memory (ROM) devices, random access memory (RAM) devices, and the like, but are not limited thereto. Other embodiments described herein relate to, for example, computer program products that may include the instructions and / or computer code described herein.

[0035] Some embodiments and / or methods described herein may be executed by software (executed 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 (executed on hardware) may be represented in various software languages (such as computer code) including C, C++, Java™, Ruby, Visual Basic™, 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, machine instructions such as those generated by a compiler, code used to generate web services, and files containing high-level instructions executed by a computer using an interpreter. For example, embodiments may be implemented using imperative programming languages (such as C, Fortran, etc.), functional programming languages (Haskell, Erlang, etc.), logic programming languages (Prolog, etc.), object-oriented programming languages (Java, C++, etc.), or other suitable programming languages and / or development tools. Additional examples of computer code include, but are not limited to, control signals, encrypted code, and compressed code.

[0036] Unless defined otherwise, 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 belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the subject matter of this specification, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the current specification, including definitions, will control. Further, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0037] The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features and advantages will be apparent from the description, drawings, and claims. [Appended Claim 1] A method for analyzing a biological sample, comprising: obtaining a biological sample attached to a first substrate; attaching a second substrate to the first substrate to form a sealed chamber on the first substrate and disposing the biological sample within an internal volume of the sealed chamber; performing a plurality of imaging cycles, each imaging cycle comprising: (a) binding a probe to the biological sample; (b) obtaining an image of the bound probe in the biological sample; (c) removing at least a portion of the probe from the biological sample and wherein the thickness of the internal volume between the first substrate and the second substrate is 250 micrometers or less. A method as described above. [Appended Claim 2] A method for analyzing a biological sample, comprising: obtaining a biological sample attached to a first substrate; attaching a second substrate to the first substrate to form a sealed chamber on the first substrate and disposing the biological sample within an internal volume of the sealed chamber; performing a plurality of imaging cycles, each imaging cycle comprising: (a) binding a probe to the biological sample; (b) obtaining an image of the bound probe in the biological sample; (c) removing at least a portion of the probe from the biological sample and wherein the thickness of the internal volume between the first substrate and the second substrate is 250 micrometers or less. The first substrate and the second substrate are substantially transparent, and an imaging window formed by the first substrate and the second substrate has a field of view of at least 500 mm 2 . A method. [Appended claim 3] A method for analyzing a biological sample, comprising the step of obtaining a biological sample attached to a first substrate, attaching a second substrate to the first substrate to form a sealed chamber on the first substrate, and disposing the biological sample within the internal volume of the sealed chamber, performing a plurality of imaging cycles, each imaging cycle comprising: (a) binding a probe to the biological sample, (b) obtaining an image of the bound probe in the biological sample, (c) removing at least a portion of the probe from the biological sample , and including wherein the first substrate is formed of glass and includes a first port defining a first channel passing through the first substrate connected to the internal volume, and a second port defining a second channel passing through the first substrate connected to the internal volume, and the closest distance between the first port and the second port measured in the plane of the second substrate is 25 mm or more. A method. [Appended claim 4] A method for analyzing a biological sample, comprising the step of obtaining a biological sample attached to a slide, contacting the slide with a gasket, the gasket including an opening, and the biological sample being disposed within the opening, and contacting the gasket with a window to enclose the biological sample within an internal volume surrounded by the slide, the gasket, and the window including The method wherein the window defines an observation area having a length of at least 25 mm and a width of at least 15 mm. [Appended Claim 5] The method according to any one of claims 1 to 3, wherein the first substrate is formed of a substantially transparent material. [Appended Claim 6] The method according to any one of claims 1 to 3, wherein the first substrate is formed of a glass or plastic material. [Appended Claim 7] The method according to any one of claims 1 to 3, wherein the first substrate includes a microscope slide. [Appended Claim 8] The method according to any one of claims 1 to 3, wherein the second substrate includes a window. [Appended Claim 9] The method according to any one of claims 1 to 3, further comprising the step of disposing a gasket between the first substrate and the second substrate. [Appended Claim 10] The method according to claim 9, wherein the gasket is formed of an adhesive. [Appended Claim 11] Before the step of attaching the second substrate to the first substrate, the step of incubating the biological sample in acetone; the step of removing residual acetone from the biological sample; and the step of attaching the second substrate to the first substrate after removing the residual acetone The method according to any one of claims 1 to 3, further comprising. [Appended Claim 12] Before the step of incubating the biological sample in acetone, the step of removing paraffin from the biological sample; and the step of exposing the biological sample to an antigen retrieval agent The method according to claim 11, further comprising. [Appended Claim 13] The method according to claim 11, wherein the biological sample is a fresh frozen sample. [Additional Item 14] The method according to claim 12, wherein the biological sample is a formalin-fixed paraffin-embedded sample. [Additional Item 15] The method according to any one of claims 1 to 3, further comprising a step of applying a dye to the biological sample before the step of performing a plurality of imaging cycles. [Additional Item 16] The method according to claim 15, wherein the dye comprises at least one element selected from the group consisting of a contrast dye, a chromogenic dye, and an immunofluorescent dye. [Additional Item 17] The method according to claim 15, wherein the dye comprises at least one of DAPI and a Hoechst dye. [Additional Item 18] The second substrate includes a first port formed by a first opening extending through the thickness of the second substrate and a second port formed by a second opening extending through the thickness of the second substrate, and a composition is introduced into the internal volume through the first port. The method according to any one of claims 1 to 3. [Additional Item 19] The method according to claim 18, wherein a minimum distance between the first port and the second port measured in a plane of the second substrate is 20 mm or more. [Additional Item 20] During each imaging cycle, a composition containing the probe is introduced through the first port, and components of the composition are removed through the second port, whereby the probe is bound to the biological sample. The method according to claim 18. [Additional Item 21] The method according to claim 18, further comprising a step of binding a capture agent to the biological sample by introducing a composition containing the capture agent through the first port before the step of performing the plurality of imaging cycles. [Additional Item 22] The method according to claim 21, wherein the capture agent comprises a binder that selectively binds to a sample component and an oligonucleotide linked to the binder. [Appended Item 23] The method according to appended item 22, wherein the binder comprises an antibody or an antibody fragment. [Appended Item 24] The method according to appended item 22, wherein the oligonucleotide comprises at least 15 nucleic acids. [Appended Item 25] The method according to appended item 22, wherein the composition comprises a plurality of different types of capture agents, and each of the different types of capture agents comprises a different type of binder that selectively binds to a different sample component and an oligonucleotide specific for each different type of binder. [Appended Item 26] The method according to appended item 25, wherein the composition comprises at least 5 different types of capture agents. [Appended Item 27] The method according to appended item 25, wherein the composition comprises at least 20 different types of capture agents. [Appended Item 28] The method according to appended item 25, wherein each of the different types of capture agents is bound to the biological sample by introducing into the internal volume of the sealed chamber a composition simultaneously comprising each of the different types of capture agents. [Appended Item 29] The method according to appended item 22, wherein the probe is an oligonucleotide comprising an oligonucleotide at least partially complementary to the oligonucleotide of the capture agent and a labeling agent linked to the oligonucleotide. [Appended Item 30] The method according to appended item 29, wherein the oligonucleotide of the probe comprises at least 20 nucleic acids. [Appended Item 31] The method according to appended item 29, wherein the labeling agent comprises a fluorescent moiety. [Appended Item 32] The step of binding the probe to the biological sample includes the step of binding one or more different types of probes to the biological sample, and each different type of probe includes different types of oligonucleotides linked to a labeling agent specific to each different type of oligonucleotide, the method according to appended claim 25. [Appended claim 33] The method according to appended claim 32, wherein the one or more different types of probes include at least three different types of probes. [Appended claim 34] The method according to appended claim 32, wherein the one or more different types of probes are simultaneously bound to the biological sample by introducing a composition comprising the one or more different types of probes into the internal volume of the sealed chamber. [Appended claim 35] The method according to appended claim 32, wherein the one or more different types of probes bind to corresponding different types of capture agents in the biological sample. [Appended claim 36] The method according to appended claim 35, wherein the one or more different types of probes hybridize to corresponding different types of capture agents in the biological sample. [Appended claim 37] The method according to appended claim 29, wherein the oligonucleotide of the probe hybridizes to the oligonucleotide of the capture agent, and the step of removing at least a part of the probe from the biological sample includes the step of dehybridizing at least a part of the probe from the capture agent. [Appended claim 38] The method according to appended claim 37, including the step of dehybridizing at least a part of the probe from the capture agent by heating the biological sample. [Appended claim 39] The method according to appended claim 37, including the step of dehybridizing at least a part of the probe from the capture agent by heating the biological sample by introducing at least one dehybridizing agent into the internal volume of the sealed chamber. [Supplementary Item 40] The method according to Supplementary Item 39, wherein the at least one dehybridizing agent contains a chaotropic agent. [Supplementary Item 41] The method according to any one of Supplementary Items 1 to 3, wherein the thickness of the internal volume is 100 micrometers or less. [Supplementary Item 42] The method according to any one of Supplementary Items 1 to 3, wherein the internal volume is 0.20 cm 3 or less. [Supplementary Item 43] The method according to any one of Supplementary Items 1 to 3, wherein the internal volume is 0.10 cm 3 or less. [Supplementary Item 44] The method according to Supplementary Item 18, further comprising connecting a port coupler to at least one of the first port and the second port before the step of introducing the composition into the internal volume of the sealed chamber. [Supplementary Item 45] The method according to Supplementary Item 44, wherein the port coupler includes a first member including at least one fluid channel aligned with one of the first port and the second port. [Supplementary Item 46] The method according to Supplementary Item 45, wherein the step of connecting the port coupler to at least one of the first port and the second port includes adhering the port coupler to the surface of the second substrate. [Supplementary Item 47] The port coupler includes a second member, and the step of connecting the port coupler to at least one of the first port and the second port includes connecting the first member and the second member of the port coupler to fix the port coupler at a predetermined position with respect to the second substrate. [Supplementary Item 48] At least one of the first member and the second member includes at least one magnet, and the first member and the second member are arranged such that the at least one magnet fixes the first member and the second member to a predetermined position with respect to the second substrate, the method according to claim 47.

Brief Description of the Drawings

[0038]

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Embodiments for Carrying Out the Invention

[0039] Like reference symbols in the various drawings indicate like elements.

[0040] General Introduction One method of multiplex staining is a method called SIMPLE, described by Glass et al., J. Histochem Cytochem. 57(10):899-905(2009). This involves immunostaining of samples using 3-amino-9-ethylcarbazole (AEC) as a detection system, mounting of coverslips, imaging by brightfield microscopy, removal of it from the microscope, removal of the coverslip, and removal of visible AEC by decolorization with ethanol. This sequence is repeated for each target of interest within the sample.

[0041] A method called t-CyCIF is described by Lin et al. in eLife 2018, 7:e31657. This involves applying up to three antibodies to the sample where each antibody is directly conjugated to a fluorescent label, applying a counterstain such as Hoechst 33342, applying a coverslip, imaging with a fluorescence microscope, removing the sample from the microscope, removing the coverslip, and bleaching the fluorophore with hydrogen peroxide and light to inactivate the dye. This sequence is repeated for each group of targets within the sample, so that the overall measurement can include many targets. Except for the final round where secondary antibodies can be used to obtain amplification, the fluorescent dyes are directly conjugated to the antibodies.

[0042] These two methods cannot be easily automated, take a long time to complete, require repeated application and removal of coverslips, and carry the risk of manual handling and sample damage. For these reasons at least, they cannot be easily deployed in settings where many samples are processed in a routine way, such as in research, clinical trials, or clinical care. However, the individual histological steps and imaging steps are flexible in that they can be performed using a wide range of equipment depending on the measurement goals of the samples involved.

[0043] CODEX® reagents, methods, and systems for sample imaging are available from Akoya Biosciences (Menlo Park, CA) and enable multiplexed sample imaging. In particular, multiple oligo-conjugated antibodies bind to multiple targets of interest using an oligo sequence unique to each antibody. Each antibody has a label consisting of an oligo-conjugated fluorescent dye designed to hybridize only with this antibody. After incubation with all primary antibodies, three groups of labels containing different dyes are introduced and hybridize specifically to the selected antibodies. The sample is imaged and each dye corresponds to a specific primary antibody. Denaturation is performed and the labels are washed away. This cycle is repeated until all targets of interest are imaged.

[0044] The CODEX® system includes a staining and imaging device. The sample is held in a glass-bottom open well on the stage of an inverted microscope. By means of automated fluidics, reagents are added to or removed from the chamber. Usually, no sample processing is performed between cycles. Also, since usually only one primary antibody incubation is used, the processing time is much shorter than the SIMPLE or t-CyCIF methods. However, during a typical experiment, neither the imaging device nor the staining device can be used for other purposes. Using an open chamber instead of a microscope slide enables automation, but a more standard sample format may be desirable. Various aspects of the methods, systems, and reagents related to the CODEX® methodology are described in U.S. Patent Nos. 10,370,698, 9,909,167, 10,017,808, 10,000,796, and 10,006,082, the entire contents of each of which are incorporated herein by reference.

[0045] Currently, as a whole, there is no method for rapidly sequential staining IHC multiplexing that can process samples provided in a standard microscope slide format without repeatedly attaching and removing a cover glass for the samples. There is also no existing method for doing this in a fully automated manner using the standard format.

[0046] The methods and systems described herein image samples in a workflow that includes multiple rounds of labeling, imaging, and label removal. The entire workflow can be performed in a semi - automated or fully automated manner. Further, the workflow can be performed using samples attached to standard microscope slides. That is, since the system can perform sample processing, imaging, and labeling, it can automate the labeling and label removal of multiple samples, and imaging can be performed with various manual or automated microscopes without sample reconstruction or cover slip manipulation between steps or cycles.

[0047] Accordingly, sequential staining multiplex IHC can be performed with very high throughput where the devices for the staining and imaging functions are operated with high utilization, and the device for one function is not idle while the other function is in progress. The system and method can include or implement a modular sample enclosure for imaging and processing samples on a microscope slide that incorporates a window through which the sample can be imaged and creates a microfluidic chamber spaced away from the surface of the sample. The chamber can include ports for introducing reagents to the sample and removing them, can be easily handled, and can be connected or disconnected to a fluid source.

[0048] In some embodiments, the sample is a tissue section or tissue microarray disposed on a microscope slide, and the window is a thin glass piece coupled to the microscope slide by a surrounding seal that holds a predetermined distance from the microscope slide. This distance can be uniform within the region forming the microfluidic chamber and can range from 40 to 250 microns. The system and method introduce and / or remove fluid reagents from the chamber formed by the window, slide, and seal. Fluid can be introduced into the microfluidic chamber through holes or openings in the front of the window, and the manifold couples the fluid from the inlet port to the holes in the window. The manifolds are preferably disposed at both ends of the window, sandwiching the sample therebetween. Due to the chamber design and flow rate, a laminar fluid flows over the sample.

[0049] The holes or openings can be arranged and sized to produce a substantially similar flow rate across all regions of the sample. This promotes uniform staining across the entire sample. This is useful for common quantitative assays, especially those incorporating amplification schemes such as hybridization and enzyme-catalyzed deposition. Also, the controlled flow is easily achieved to expose the sample to fresh reagent material at a predictable rate when the sample is depleted or used up. In some embodiments, the holes at one end of the window form equally spaced arcs between the holes, and all the holes are equidistant from the input ports of the associated manifold. This produces a uniform sheet flow across the entire sample. The same arrangement can be used for both manifolds.

[0050] In some embodiments, the substrate holds a plurality of samples, and each sample is on its own microscope slide with a microfluidic chamber enclosure with a window, so that multiple samples can be efficiently processed in groups.

[0051] The ports formed in the manifold can couple fluid into or out of the chamber from a pipette tip, or a tube, or both, or from the chamber into a pipette tip, or a tube, or both. Where tubes are present, quick connect fixtures or other fittings can be incorporated to easily connect the chamber to an automated fluid assembly and / or a manual pipette, or to remove the chamber from the connection.

[0052] During sample processing within the sealed chamber, the reagent remains in contact with the sample for an extended period of time, enabling incubation of the sample. The sample during incubation can be easily processed or transferred without disturbing the ongoing incubation.

[0053] The device of the present invention can generally be used over a wide range of temperatures, typically from -4°C to 45°C, and any smaller temperature range within this range. The internal volume of the sealed chamber is relatively small, for example, in the case of a 4×2 cm window with a spacing from the window to the slide of about 50 micrometers, it can be about 40 microliters. The dead volume within the sealed chamber can be made 5 microliters or less, reducing the reagent volume and cost. More importantly, by achieving a very small dead volume, the residual amount of a particular reagent remaining in the chamber after the removal step is very small, ensuring that cross - contamination between steps is greatly eliminated.

[0054] Typically, the sealed chambers described herein are practical for use in a wide range of environments such as academic research, translational studies, testing, clinical care, etc. because they are inexpensive to manufacture and easy to use.

[0055] The methods described herein include multiplex labeling and imaging of samples. Some methods relate to multiplex labeling and imaging of formalin - fixed paraffin - embedded samples on microscope slides. Other methods relate to labeling and imaging of fresh frozen samples.

[0056] To form a chamber for encapsulating a 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 with a hole or opening in the second substrate, separated from the first substrate by a selected distance. Reagents are introduced, removed, and moved across the sample surface by directing the reagents to flow through these holes or openings. These reagents are used to label components (such as molecular targets) in the sample, and the labels are imaged from the window using a microscope. After one or more labels are removed, erased, or both, the cycle of labeling, imaging, and removal or erasure is repeated. In this way, a multiplexed image of the sample is obtained. This image can be analyzed to gain insights about the sample or the organism from which the sample was extracted.

[0057] In some embodiments of this type, the sample is incubated with one or more primary antibodies before the fluid chamber is formed by attaching the second substrate to the first substrate. In other embodiments, the incubation with the primary antibody is performed within the chamber after it is formed.

[0058] Continuous staining and multiplex imaging of biological samples can be performed using various methods. These methods include, but are not limited to, labeling the bright field with 3-amino-9-ethylcarbazole (AEC), where the label is erased using ethanol; directly labeling with an antibody conjugated to a fluorescent dye, where the label is erased using hydrogen peroxide; indirectly immunofluorescently labeling the primary antibody, where the fluorescent label is erased by hydrogen peroxidase or removed by removing the primary antibody, secondary antibody, or both; labeling with an oligo-binding fluorescent dye that hybridizes to the corresponding oligo sequence bound to the antibody, where the label is removed by dehybridization.

[0059] This method also includes staining with histochemical dyes such as eosin, hematoxylin, 4′,6-diamidino-2-phenylindole (DAPI), periodic acid Schiff staining (PAS), methylene blue, Hoechst staining (Hoechst 33342, Hoechst 34580, Hoechst 33258, etc.), which are removed by ethanol or other solvents. These can often provide information regarding the presence, location, and state of various intracellular compartments that are useful and complementary to molecular IHC information.

[0060] This method can also include fluorescence multiplexing with amplification. In one embodiment, the primary antibody is conjugated to a unique oligo sequence, and for labeling, an enzyme catalyst such as horseradish peroxidase (HRP) conjugated to the corresponding oligo sequence is used. One labeled species is introduced into the device holding the sample, hybridizes with the corresponding antibody, and tyramide signal amplification is used to deposit a fluorescent dye. The label is then dehybridized and washed away. This process can be repeated several times with different dyes having different spectra, and the sample is imaged. Optionally, the dyes can be inactivated later by using hydrogen peroxide and light.

[0061] In some embodiments, rolling circle amplification (RCA) can be used to amplify the probes, or linear and / or branched oligo structures can be used to attach multiple dye molecules per labeled antibody in one or more cycles of labeling and imaging.

[0062] Furthermore, any of the aforementioned methods can be combined. For example, the methods for processing and analyzing the sample can include one or more stainings, imaging, and removals with histochemical dyes, as well as one or more IHC labelings, imaging, and removals or erasures of the labels.

[0063] The chamber surrounding the sample can remain in a predetermined position over multiple imaging and labeling cycles. This is beneficial because the workflow is practical for processing large batches of samples or in an automated manner. Also, the sample is not vulnerable to damage from handling procedures such as window attachment and removal.

[0064] 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. Thus, since information about the sample emerges during processing, image acquisition can be adjusted to the sample. Due to the modularity of sample processing, it is practical to isolate the sample after the IHC cycle used for target selection is completed until the selection of the sample components to be imaged in subsequent cycles is made, after which sample processing resumes.

[0065] Sample enclosure As described above, the sample enclosure described herein is generally implemented by attaching one or more components to a substrate that supports a biological sample. For this purpose, a variety of different components can be used. FIG. 1A is a schematic diagram showing an exploded view of the components used to form one example of a sample enclosure. The first substrate 150 supports a biological sample 151 (e.g., a tissue section). To form an enclosure 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. Attaching the second substrate 101 to the first substrate 150 using the gasket 102 creates a sealed chamber having 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 opening region 113 of the gasket 102.

[0066] The second substrate 101 includes two or more ports to enable the flow of fluid into and out of the internal volume of the chamber. Generally, each port is formed as an opening that extends through the second substrate 101 and connects to the internal volume of the sealed chamber. The second substrate 101 may have one or more inlet ports (e.g., two or more, three or more, four or more, five or more, or more). In FIG. 1A, four inlet ports 103a - 103d are shown as an example. 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 more). In FIG. 1A, four outlet ports 104a - 104d are shown as an example.

[0067] The inlet manifold 105 can be attached to the second substrate 101 via an adhesive layer 121 having openings aligned with the ports 103a - 103d, if necessary. The fluid flowing into the inlet port 107 of the inlet manifold 105 is transported through the internal manifold channels to the ports 103a - 103d, thereby introducing the fluid into the internal volume of the sealed chamber. The outlet manifold 110 can optionally be attached to the second substrate 101 via an adhesive layer 123 having openings aligned with the ports 104a - 104d. The fluid flowing out of the internal volume of the sealed chamber is transported through the outlet ports 104a - 104d to the manifold 110, where it is discharged from the outlet port 112.

[0068] As described above, when assembled, the aforementioned components form a sealed chamber around the sample 151, which is disposed within the internal volume of the chamber. Reagents, compositions, and other fluids (and fluid support compounds) can be introduced into the internal volume to contact, react with, bind to, or otherwise interact with the sample 151. Fluids, components of the composition, and reagents can 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 enclosure.

[0069] In FIG. 1A, 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 certain embodiments, the thickness of the internal volume of the sealed chamber is at least partially determined 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).

[0070] In particular, within the range of thicknesses from 30 micrometers to 250 micrometers, laminar fluid flow is promoted, intimate contact between the raw reagent and the sample is ensured, and thus it has been observed that the sample processing time is reduced compared to processing with more turbulent fluid flow. Further, since the internal volume of the sealed chamber is relatively small compared to the volume of reagents used in bench-top processing and automated staining devices, reagent consumption is reduced compared to these techniques. Thus, sample processing can be done faster and / or more economically compared to these alternatives.

[0071] In some embodiments, the internal volume of the sealed chamber is 0.40 cm 3 or less (e.g., 0.35 cm 3 or less, 0.30 cm 3 or less, 0.25 cm 3 or less, 0.20 cm 3 or less, 0.15 cm 3 or less, 0.10 cm 3 or less, 0.08 cm 3 or less, 0.07 cm 3 or less, 0.06 cm 3 or less, 0.05 cm 3 or less, 0.04 cm 3 or less, 0.03 cm 3 or less, 0.02 cm 3 or less, 0.01 cm 3 or less).

[0072] In FIG. 1A, either 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 at any wavelength from 450 nm to 650 nm is absorbed or reflected by the substrate. When substrates 150 and 101 are substantially transparent and manifolds 105 and 110 are disposed at the ends of substrate 101, the field of view of the sealed chamber is defined by the opening region 113 of gasket 102 in FIG. 1A. Alternatively, in some embodiments, other components used to form the sealed chamber (one or more manifolds, substrates 101 and / or 150, and optional spatial filtering layers, etc.) define the field of view of the sealed chamber.

[0073] As used herein, the "field of view" of the sealed chamber is the area of the largest rectangular or square substantially transparent window through which a sample on substrate 150 can be imaged. The field of view is represented by the dashed rectangle 115 in FIG. 1A. Note that the field of view may 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 350 mm 2 , at least 400 mm 2 , at least 450 mm 2 , at least 500 mm 2 , at least 550 mm 2 , at least 600 mm 2 , at least 650 mm 2 , at least 700 mm 2 , at least 750 mm 2 ).

[0074] As used herein, the "observation region" of the sealed chamber is the region of a substantially transparent window through which the sample on the substrate 150 can be imaged. The observation region can be larger than the field of view. The length of the observation region is the maximum dimension of the observation region in a direction parallel to one side of the substrate 150, and the width of the observation region is the maximum dimension of the observation region in a direction orthogonal to the length, and is smaller than the length. Each length (L VA ) and width (W VA ) of the observation region are shown in FIG. 1A.

[0075] In some embodiments, the length of the observation region is 20 mm or more (for example, 25 mm or more, 30 mm or more, 35 mm or more, 40 mm or more, 45 mm or more, 50 mm or more, 55 mm or more, 60 mm or more, 65 mm or more, 70 mm or more). In certain embodiments, the width of the observation region is 10 mm or more (for example, 15 mm or more, 20 mm or more, 25 mm or more, 30 mm or more).

[0076] The substrate, gasket, ports, and manifold can be sized and positioned to create a laminar flow of reagent over the sample 151 on the substrate 150. Thus, the sample 151 can be labeled, imaged, and unlabeled within the internal volume of the sealed chamber. To ensure a relatively uniform staining or labeling, the flow of reagent to the sample 151 may be relatively similar at all locations on the sample surface. This is particularly important during processing that is strongly dependent on reagent concentration and time, or during processing where the reagent is depleted by interaction with the sample, or during processing that normally does not reach saturation but instead produces results that depend on the reaction rate. An example of such a process is the IHC labeling amplification step.

[0077] Nearly uniform flow of the reagent or composition across sample 151 can be achieved in several ways. For example, in a particular embodiment shown in FIG. 1A, manifold 105 includes a plurality of flow channels coupled to a plurality of input ports 103a-103d, and the impedance to fluid flow along the fluid flow path defined by the flow channels and input ports is substantially the same. Input ports 103a-103d are arranged along an arc within a sealed chamber to assist in impedance matching, and substantially equal flow rates are supplied through the ports. Due to the position and dimensions of the chamber and ports, the flow is laminar at the flow rates used for sample processing. The fluid introduced as individual streams at the input ports forms a continuous sheet of flow within a few millimeters after entering the chamber. As a result, the sample is processed uniformly with a minimal reagent gradient.

[0078] In some embodiments, sample processing includes flowing a series of reagents over the sample in sequence. When a new reagent is introduced into the input port, the fluid enters a front chamber whose shape is determined by the port arrangement. Here, the term "front" is used to indicate a depiction where the contents of the chamber change from one reagent to the next.

[0079] In this example, the port arrangement causes the front to form an arc. Each time a new reagent is introduced, this front moves along the slide and moves from one manifold to another. Typically, at least a sufficient amount of new reagent is introduced to ensure that the front completely passes through the sample region. Since laminar flow occurs within a sealed chamber, the discharge of the previous reagent is rapid, and the number of fluid exchanges required to produce a complete turnover of the reagent is less compared to other slide-based sample processing approaches. In some cases, all that is required is to advance the front beyond the sample region. In other cases, several times the amount of new reagent is introduced so that not even a trace amount of the previous reagent remains in the sample region.

[0080] Generally, the second substrate 101 can include one or more inlet ports 103 (e.g., two or more, three or more, four or more, five or more, or more). Further, the second substrate 101 can include one or more outlet ports 104 (e.g., two or more, three or more, four or more, five or more, or more). Generally, the inlet port 103 is disposed near the first side of the second substrate 101, and the outlet port 104 is disposed near the second side of the second substrate 101 on the opposite side of the first side. Typically, the inlet and outlet ports are disposed on opposite sides of the second substrate 101 such that the fluid introduced through the inlet port 103 and removed through the outlet port 104 flows over the surface of the sample 151 during transport between the ports.

[0081] In some embodiments, as shown in FIG. 1A, the minimum or closest distance p between any one of the inlet ports 103 and any one of the outlet ports 104, measured in the plane of the second substrate min is 20 mm or more (e.g., 25 mm or more, 30 mm or more, 35 mm or more, 40 mm or more, 45 mm or more, 50 mm or more, 55 mm or more, 60 mm or more, 65 mm or more, or more). If 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.

[0082] The first substrate 150 can be formed from a variety of 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 from a variety of 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 a piece of Corning Eagle XG glass (Corning, NY) with a thickness of 0.5 mm, implemented as a piece in the range of 24×55 mm.

[0083] The first substrate 150 can generally 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 can generally 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 0.3 mm or less, 0.25 mm or less, 0.2 mm or less, 0.1 mm or less).

[0084] Generally, manifolds 105 and 110 can be formed from a variety of materials including plastics, rubber-based materials, and metals. In some embodiments, for example, manifolds 105 and / or 110 are formed of polyetheretherketone (PEEK) and are solid printed to provide the ports and internal pipes that connect them.

[0085] Gasket 102 is generally implemented as a pressure-sensitive adhesive selected for good adhesion and chemical compatibility with the reagents, cleaning agents, and other fluids used in the process. In embodiments where the thickness of the gasket determines the thickness of the internal volume of the chamber, the selection of the gasket provides a way to select the thickness of the chamber to a desired value. In some embodiments, for example, gasket 102 can be formed from an acrylic material such as 3M adhesive transfer tape 966 (3M, St. Paul, Minn.), although in other situations other materials may be preferred depending on the desired thickness and the chemical nature of the reagents used in the process.

[0086] The adhesive layer 121 and / or 123 can generally correspond to a pressure-sensitive adhesive as described above. Alternatively, other adhesives such as epoxy or Master Bond EP21LV (Hackensack, New Jersey) can be used. Generally, the adhesive layer provides a liquid-tight seal between the manifold and the second substrate without impeding the flow of fluid. Selection criteria include chemical compatibility with the reagents used in sample processing and adhesion to the surface of the manifold associated with the glass.

[0087] Other techniques can be used to generate a uniform flow of various shapes. For example, the manifold can have channels of different lengths with a diameter selected to achieve approximately equal flow rates / volumes at the inlet ports.

[0088] The terms input port and output port are used above for clarity of explanation, and the fluid flow can potentially be completely or mainly in one direction. However, the closed chamber can be operated with fluid flowing in either direction if beneficial for a particular sample processing operation. For example, the flow can be circulated in either direction to achieve an effect such as that achieved by rocking agitation during bench processing, i.e., promoting the interaction between the sample and the reagent. Also, such an action reduces the extent to which local sample reactions deplete the reagents near them by circulating which reagent amounts interact with which sample regions.

[0089] In Figure 1A, the manifolds 105 and 110 provide a single opening or port for fluid input and output. This can be coupled to a tube by pushing a short length of a #19 gauge stainless steel hypodermic needle into the port and attaching the tube to the protruding portion of the stock using friction fitting to a tube of appropriate size. The tube can include a quick connect fitting at the other end if desired.

[0090] Alternative connection schemes are possible where the manifold 105 and / or 110 incorporates an elastomeric seal such as an O-ring to make direct contact with the pipette input and / or output ports 107 and / or 112. This is advantageous in situations where reagents are limited or costly as it minimizes dead volume. The overall arrangement is very efficient with respect to reagent use as the volume required to fill the chamber can be very small. In some embodiments, for example, the overall dead volume within the sealed chamber is 5 microliters or less (e.g., 4 microliters or less, 3 microliters or less, 2 microliters or less, 1 microliter or less).

[0091] Due to the possibility of various different connections between the manifold and external fluid devices, the same manifold can be connected to a pipette or press-fit tube at different times. In this way, the same sealed chamber can be easily connected to a manual fluid item such as a pipette or an automated fluid device via a tube or other arrangement.

[0092] An example of a sealed chamber is shown in FIG. 1A. However, the sealed chambers described herein can be implemented in various ways. FIG. 1B shows a top view of another example of a sealed chamber formed by a first substrate 150, a gasket 102, and a second substrate 101 (the first substrate 150 is not shown in FIG. 1B for clarity). As shown in FIG. 1B, the chamber includes a single input port 103 and a single output port 104, which are each formed as openings in the second substrate 101 that extend through the thickness of the substrate. The opening region 113 of the gasket 102 is shaped as an irregular hexagon (i.e., a hexagon with non-uniform side lengths). Ports 103 and 104 are located at opposite vertices of the hexagonal opening along the central axis of the gasket. The gasket 102 of FIG. 1B can have any one or more of the attributes discussed above in relation to the gasket 102 of FIG. 1A.

[0093] Manifolds 105 and 107 may also be referred to as port couplers, especially when the sealed chamber includes a single input port and / or a single output port. In addition to manifolds 105 and 107, other implementations of port couplers can also be used. For example, FIG. 1C shows a schematic diagram of another example of port coupler 160. Port coupler 160 includes a first member 170 and a second member 172. Magnet 176 is embedded within the body of the first member 170 (optionally, the magnet may be embedded within the body of the second member 172 as an alternative to, or in addition to, the first member 170). Member 172 is formed of a magnetic material such as stainless steel. The first member 170 includes an opening 174 that connects to port 103 or port 104 (FIG. 1B). Within the first member 170, the opening 174 is connected to port 178 via an internal channel, and port 178 is then connected to fluid conduit 180.

[0094] To engage port coupler 160 with the second substrate 101, the first member 170 is positioned on top of the second substrate 101 using the opening 174 aligned with port 103 or 104. The second member 172 is positioned on the opposite side of the first member 170 below the first substrate 150. The magnetic force between magnet 176 and the second member 172 ensures that the port coupler 160 remains fixed in place.

[0095] To fix members 170 and 172 to the second substrate 101, various other locking mechanisms can also be used. For example, in some embodiments, mechanisms such as clips, latches, pins, tabs, and keys can be used to lock members 170 and 172 in place relative to each other and to the second substrate 101. As another alternative, members 170 and / or 172 can be affixed to the second substrate 101 using an adhesive such as any of the aforementioned adhesives. When using an adhesive, the second member 172 can be removed if necessary.

[0096] In some embodiments, the seal member or layer is disposed between the port coupler 160 and the second substrate 101 to ensure that a fluid-tight seal is formed between the opening 174 and the port 103 or 104. In some embodiments, as shown in FIG. 1C, an elastomeric member such as the seal ring 175 is disposed on the first member 170 or the second substrate 101. The elastomeric member 175 is compressed between the first member 170 and the second substrate 101 to form a seal. Alternatively, in certain embodiments, an elastomeric sheet (similar to layer 212 in FIG. 1A) is disposed on the first member 170 or the second substrate 101 and compressed between the first member 170 and the second substrate 101 to form a seal.

[0097] Sample preparation method As described above, the sealed chamber described herein can be formed on a substrate that supports various samples. Examples of such samples include, but are not limited to, fresh tissue such as biopsy sections, fresh frozen (FF) tissue sections, and formalin-fixed, paraffin-embedded (FFPE) tissue sections. The methods described herein are generally used to perform one or more cycles of immunohistochemical (IHC) staining and imaging of a biological sample enclosed within a chamber formed as described above.

[0098] FIG. 2 is a flowchart showing an example series of steps for labeling and imaging an FF tissue section. 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 warming the tissue section under ambient conditions. Next, in step 204, the tissue section is fixed, for example, by incubating the tissue section in an acetone bath at room temperature for 10 minutes. Next, in step 206, the tissue section is dried by evaporating the residual acetone in the tissue section either passively or by heating.

[0099] Next, in step 208, the tissue is rehydrated in a solution (e.g., in a solution such as potassium buffered sodium (PBS) for several minutes), and then, in step 210, a tissue blocking step is performed. Next, in step 212, the tissue section is incubated with one or more capture agents, each of which contains a primary antibody that specifically binds to a component (e.g., a target) in the sample. Following the incubation with the primary antibody, the tissue section is washed in step 214, and in step 216, initial fixation is performed with an aqueous paraformaldehyde (PFA) solution.

[0100] Next, the tissue section is washed in step 218 and undergoes a second methanol fixation step 220. After washing again in step 222, the tissue section undergoes a bovine serum albumin (BSA) fixation step 224. Finally, the sample is washed again in step 226 and either sent to a storage location in step 228 or subjected to one or more labeling and imaging cycles 230.

[0101] Within the exemplary steps shown in FIG. 2, the sealed chamber can be formed as described above at any point in the workflow after step 206. That is, at any time after the residual acetone has been removed from the tissue section in step 206, the sealed chamber can be formed, and 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) from one or more output ports 104.

[0102] FIG. 3 is a flowchart showing an example series of steps for labeling and imaging an FFPE tissue section. In a first step 302, the tissue section is deparaffinized using any of several well-known deparaffinization protocols, e.g., using sequential xylene and ethanol washes.

[0103] Next, in step 304, antigen retrieval (AR) is performed. There are many techniques for AR, and the selection of an appropriate technique depends on factors such as the type of tissue, fixation conditions, and target epitope detected by the IHC label. For example, conventional techniques of AR that can be used include heat-induced epitope retrieval (HIER) and proteolysis-induced epitope retrieval (PIER). These techniques can be performed using the sample 151 on the first substrate 150 (e.g., on a microscope slide) as in the conventional practice. Following AR, the tissue section is washed (e.g., with a PBS solution) to remove residual AR reagents and then dried in step 306.

[0104] Subsequent steps 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, and 330 are the same as the corresponding steps 208 - 230 above, so the description of these steps will not be repeated.

[0105] Within the exemplary steps shown in FIG. 3, the sealed chamber can be formed as described above at any point in the workflow after step 302. That is, at any time after deparaffinization, the tissue section can be rinsed with ethanol, acetone, toluene, or another non-aqueous solvent, and the residual solvent can be removed from the sample by drying to form the sealed chamber. 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.

[0106] Sample analysis workflow The above preparation steps can be used as precursors to a variety of different labeling and imaging workflows corresponding to steps 230 and 330 of FIGS. 1 and 2. Note that in steps 228 and 328, the prepared sample can be delivered to a storage facility within the sealed chamber formed as described above. For example, the sample can be stored at a low temperature (e.g., about 4° C.) and removed from the storage location at a later date (e.g., more than 1 hour, several days, weeks, months, or more after preparation) and warmed to room temperature for labeling and imaging. In some embodiments, the prepared sample can go through one or more labeling and imaging cycles and then be sent (or returned) to a storage location in a storage facility or the storage location. The same sample within the sealed chamber can then be retrieved for additional labeling and imaging cycles. The sample can be sent and retrieved to storage multiple times, and some or all of the labeling and imaging steps can be repeated after intervals of sample storage.

[0107] Furthermore, after completion of the labeling and imaging cycles (and, in some embodiments, after retrieving the sample again from the storage location), the chamber surrounding 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, the sample 151 can be subjected to genetic analysis where the sample cells are lysed, DNA and / or RNA are collected, and the collected nucleic acids are sequenced using any of a variety of different next-generation sequencing (NGS) techniques.

[0108] After checking the images obtained after one or more cycles of labeling and imaging, and repeating one or more steps of labeling and imaging (e.g., to verify imaging parameters such as exposure time and / or to detect weakly expressed sample components), provides significant advantages especially compared to conventional IHC methods where samples are generally not re-labeled and imaged after being stored for a certain period. Further, by intermittently storing the sample and then acquiring and resuming the labeling and imaging cycles, the user can schedule the analysis according to the lab's schedule and priorities.

[0109] In steps 230 and 330, various different labeling and imaging workflows can be used. Each workflow can include a single step of labeling and imaging, or multiple steps of labeling and imaging (e.g., multiple cycles of labeling and imaging). Examples of these various types of workflows are described below.

[0110] Non-sequential IHC In some embodiments, a single round of labeling and imaging of the biological sample within the sealed chamber is performed. The label attached to the sample can be removed, but may also remain on the sample after imaging. To label the sample, a composition containing one or more labels or dyes (e.g., a fluid-based composition) is delivered to the sealed chamber via one or more ports 103, and the label or dye interacts with the sample relatively quickly after introduction compared to conventional methods of sample processing. The composition can be delivered by an automated fluid assembly or introduced manually by a technician.

[0111] In some embodiments, the composition delivered to the sealed chamber comprises a single type of label or stain that binds to a specific type of component (e.g., target) in the sample, such as a specific antigen or expressed protein marker. In certain embodiments, the composition delivered to the sealed chamber comprises a plurality of different types of labels or dyes, each of which specifically binds to a different type of component (e.g., target) in the sample.

[0112] For example, in some embodiments, each of the different types of labels can comprise a primary antibody or an antibody fragment conjugated to a fluorescent label. The primary antibody or antibody fragment binds to a specific component (e.g., target antigen or protein) in the sample. By exposing the label to incident light and acquiring an image of the fluorescence emission from the label, the spatial distribution and quantitative amount of the corresponding component in the sample can be determined.

[0113] In some embodiments, tyramide signal amplification (TSA) can be used to label the sample for imaging. For the component of interest in the sample, a first composition is delivered to the sample that comprises a primary antibody or antibody fragment that specifically binds to that component. Next, a second composition comprising a secondary antibody that specifically binds to the primary antibody is introduced. The secondary antibody is conjugated to an enzyme catalyst (e.g., horseradish peroxidase (HRP)). Next, a third composition comprising tyramide labeled with a label is introduced, and the enzyme catalyst catalyzes the conversion of the tyramide to a tyramide radical, which then binds to tyrosine residues proximal to the position of the primary antibody. In this way, significant amplification of the fluorescent 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 content of which is incorporated herein by reference.

[0114] In some embodiments, instead of a direct bond between the secondary antibody and the enzyme catalyst, the secondary antibody can be biotinylated and the enzyme catalyst (e.g., HRP) can be bound to streptavidin. The secondary antibody is first delivered to the sample, followed by the enzyme catalyst selectively binding the catalyst to the secondary antibody, and then the TSA proceeds as described above.

[0115] In certain embodiments, one or more dyes can be applied to the sample. Staining can be applied before, during, or after one or more cycles of labeling and imaging according to standard staining protocols. The dye can be applied to the sample by delivering the staining solution to a sealed chamber or by applying the dye to the sample before forming the sealed chamber. A variety of different stains can be used, including but not limited to DAPI, Hoechst staining, hematoxylin, eosin, and combinations of multiple stains.

[0116] In all of the foregoing workflows, images of the labeled sample and the dye are acquired using an image detector such as a CCD camera or a CMOS detector. Incident radiation from a radiation source is directed at the stained / labeled sample, and the light emitted from the sample is detected (e.g., imaged) by the detector. In some embodiments, when the sample includes an absorptive (e.g., chromogenic) stain such as a contrast stain, the light emitted from the sample generally corresponds to the incident light transmitted through or reflected by the sample. When the sample includes a fluorescent stain (e.g., immunofluorescent stain), the light emitted from the sample generally corresponds to the fluorescent emission from the stain in response to the incident radiation.

[0117] When the sample includes a fluorescent label (e.g., a fluorescent moiety that specifically binds or is associated with a particular component (target) in the sample), the incident radiation causes the label to emit fluorescence that is imaged by the detector. The relative amount of the component (target) can be quantitatively determined at each position within the sample using the amount and spatial distribution of the fluorescence emission.

[0118] Examples of chromogenic dyes that can be used include, but are not limited to, xanthene-based dyes such as fluorescein dyes and / or rhodamine dyes. Examples of suitable fluorescein and rhodamine dyes include, but are not limited to, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (commonly 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 (R6G5 or G5), 6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110. Chromogenic statins can also include cyanine-based dyes. Suitable examples of such dyes include, but are not limited to, the dyes Cy3, Cy5, and Cy7. Chromogenic dyes can be coumarin dyes (such as umbelliferone), benzimidazole dyes (such as any of the Hoechst dyes, e.g., Hoechst 33258), phenanthridine dyes (such as Texas Red), ethidium dyes, acridine dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethine dyes (such as any of the BODIPY dyes), and quinoline dyes.

[0119] Examples of immunofluorescence staining that can be used include, but are not limited to, pyrene, coumarin, diethylaminocoumarin, FAM, fluorescein chlorotriazinyl, fluorescein, R110, JOE, R6G, tetramethylrhodamine, TAMRA, Lissamine, naphthofluorescein, Texas Red, Cy3, and Cy5.

[0120] Sequential multiplex IHC In addition to a single round of simple or multiplexed IHC labeling, multiplexed IHC labeling and imaging by sequential labeling and imaging cycles can be implemented. In the workflow shown in Figure 2, an oligo-labeled multiplex analysis is performed. In this type of analysis, there is a single incubation with the primary antibody, followed by a round of oligo-mediated labeling by hybridization, imaging of the label, dehybridization, and removal of the label by a washing step.

[0121] The sample can be subjected to acetone fixation and evaporation. Subsequently, a sealed chamber is formed with the sample within the internal volume of the chamber. The primary incubation step can be performed by direct pipette injection into the sealed chamber. Fixation and periodic labeling, imaging, and label removal can be performed by an automated fluid system such as that used in the Akoya CODEX (registered trademark) platform (Akoya Biosciences, Menlo Park, CA) connected to the input port 103 and output port 104 of the chamber.

[0122] After each round of labeling, the sample can be imaged using a microscope equipped with an automated stage. For example, the microscope can use a Nikon CFI S Plan Fluor ELWD20XC with an NA of 0.45. Its focus color can be set to correct for the window and the thickness of the fluid layer between the sample and the objective lens. This correction can be performed by adjusting before the start of the staining or by any method, such that the objective lens is configured for proper imaging with the actual material and thickness present during successive imaging steps. Alternatively, other objectives can be used, including but not limited to a Nikon CFI S Plan Fluor LWD 20XC with an NA of 0.70.

[0123] Generally, the sealed chambers described herein can be used in any workflow that enables periodic labeling, imaging, and removal of labels from a sample. Methods that include these steps after a single primary incubation are particularly advantageous because they reduce the analysis time resulting from introducing all primary antibodies to the sample in a single incubation step. However, it is also possible to implement methods that include multiple incubation steps with primary antibodies (e.g., interleaved between labeling and imaging cycles), similar to methods without individual primary antibody incubations, after additional labeling has been performed prior to imaging.

[0124] In certain embodiments, following the incubation with primary antibodies, one or more labeling and imaging cycles can be performed, with label amplification occurring when the sample is labeled. A variety of different methods can be used for amplification, including rolling circle amplification (RCA), branch or ligation amplification (BLA), oligo-conjugated enzyme amplification such as tyramide signal amplification (TSA) using oligo-conjugated HRP, and oligo-mediated labeling incorporating combinations of these techniques.

[0125] One or more labeling and imaging cycles can also be performed without label amplification. Additionally, in some embodiments, the first cycle of labeling, imaging, and label removal is performed using one type of label, such as a non-amplified oligo label, and additional cycles are performed (either immediately or after additional cycles) based on amplified labeling such as RCA, BLA, TSA. In other embodiments, the cycle order is reversed.

[0126] In certain embodiments where one or more labeling and imaging cycles are performed, label removal may be omitted in any cycle, such as the final cycle. This saves time and effort, and the sample remains in a state where it can potentially be imaged again in the future with the last applied label present. Note that the TSA reaction deposits compounds on the sample that are not as easily removed from the sample as some of the other workflows described herein, and the compounds may interfere with subsequent labeling and / or imaging. Thus, in some embodiments, it may be advantageous to use a TSA-based labeling reaction after other cycles are complete.

[0127] In some embodiments, one or more samples, each enclosed in a corresponding chamber, are processed by a system comprising an automated labeling station with a fluid assembly and an automated imaging station with an image detector. The system can perform primary incubation and sample fixation steps as needed. Alternatively, these steps can be performed manually by a technician. The system performs one or more imaging cycles (e.g., labeling, imaging, label removal) in a fully automated manner without user intervention. When analyzing multiple samples, the fluidic operations and imaging operations can be interleaved between samples to improve efficiency. For example, when analyzing four samples, the first sample can receive an automated incubation with a primary antibody, the system can deliver a probe to the second sample as part of the labeling and imaging cycle, the system can acquire an image of one or more labeling agents introduced into the third sample as part of the labeling and imaging cycle, and the system can deliver a label removal agent to the fourth sample as part of the labeling and imaging cycle.

[0128] IHC and histological staining In some embodiments, one or more IHC cycles (e.g., labeling and imaging cycles) can be performed, after which histological staining of the sample is performed. Alternatively, histological staining can be performed before performing one or more IHC cycles (e.g., labeling and imaging cycles) on the sample.

[0129] A wide variety of histological dyes can be used in such workflows. For example, by combining histological staining with one or more labeling and imaging cycles, the sample can be imaged as stained with hematoxylin and eosin (H&E), and can also be imaged using one or more IHC probes applied and imaged successively on the same sample. This allows a pathologist to identify, select regions for analysis, and generally visualize the structure in the same sample from which quantification of components (i.e., biomarker data) has been obtained.

[0130] Dyes localized in the selected tissue region or material may be useful for image processing such as membrane localization staining and may be useful for automated analysis of IHC images of the same sample. For example, an image corresponding to histological staining can function as input data for a cell segmentation algorithm.

[0131] Efficiency of the workflow The sealed chambers described herein allow for repeated staining and imaging of slide-supported samples. Further, samples within the sealed chamber can be easily processed without exposing the samples to danger and can be easily connected to manual or automated fluid assemblies and devices.

[0132] The sealed substrate can be placed in a carrier, which is convenient for efficiently processing a large number of samples. For example, a carrier equipped with four or more such substrates can be connected to an automated fluid device using an interlocking fluid connection. Once labeling is complete, the samples can be detached and placed manually or by robot onto an automated imaging device. Once scanning is complete, the batch of substrates can be reconnected to the fluid device, the labels removed, and the next cycle of labeling introduced. This cycle can be repeated to perform multiplexed IHC analysis in sequential labeling and imaging cycles.

[0133] Such a workflow has several advantages. First, multiple samples can be processed in each processing step, whether manually or automatically. Second, while a given carrier with one group of samples is connected to the automated fluid assembly, the automated imaging station or device can scan another carrier with a different group of samples. While one is active, neither the imaging nor the fluid assembly is idle, achieving maximum utilization.

[0134] Furthermore, the samples are not connected to the fluid elements during imaging. This reduces or eliminates the risk of leakage or spillage of the imaging components. Additionally, the methods described herein can be performed using a variety of imaging stations (e.g., microscopes). This is because the imaging station does not need to be tightly integrated with the fluid assembly to be a closed system. The second substrate and fluid within the microfluidic chamber covering the sample have a "focus collar" to compensate for various amounts of glass or fluid in the optical path and have optical effects and objective lenses that can be used to acquire a sample image.

[0135] The same sample can be imaged by several different imaging stations or systems within a given imaging cycle, or between different imaging cycles, in an analysis that includes one or more labeling and imaging cycles. For example, one or more sample images can be acquired using special microscopy techniques such as confocal, multiphoton, or super-resolution techniques to obtain high-resolution images (and high-resolution quantitative information regarding biomarker expression) at locations where important sample components (targets) are present. In other cycles, the sample can be imaged at low resolution and / or with various imaging stations or systems such as whole slide scanners.

[0136] Guided Labeling and Imaging The methods described herein can be used to implement a guided labeling and imaging workflow. As an example, after a sealed chamber is formed around the sample, histological staining can be applied to the sample or a first labeling and imaging cycle can be performed using one or more different types of probes. Based on the characteristics of the sample (at least in part, determinable from one or more images of histological staining and / or one or more labeling agents corresponding to probes within the sample), a technician or an automated controller executing the analysis software determines whether to further analyze the sample. In this way, the suitability of each sample can be confirmed, at least in part, based on its histological and / or IHC response, before the cost and time of further analysis are expended. Degraded or unusable samples can be identified at this point.

[0137] In some embodiments, the sample information obtained by the methods described above is used by a technician or an automated controller to select, for further sample analysis, one from among a plurality of possible staining / labeling and imaging protocols. Samples from a patient or other subject can be sent along one of several courses of staining / labeling and imaging, depending on the information initially obtained.

[0138] Alternatively, the sample can be processed through one or more cycles of IHC labeling (e.g., labeling and imaging cycles). Unexpected or unforecastable results obtained in this way may indicate that further IHC measurements of one or more other markers are valuable in order to more fully understand the sample. To obtain these measurements, additional steps can be performed that include any of the steps described herein, such as incubation with one or more primary antibodies, labeling, and imaging.

[0139] Following one or more cycles of IHC labeling and imaging, the information obtained from the sample image can be used to evaluate the labeling quality and imaging quality obtained. If these are found to be insufficient, the sample can be reprocessed through one or more labeling and imaging cycles. Such an evaluation can be performed after each labeling and imaging cycle, or at the end of all such cycles, and the sample image can be evaluated by a technician or by an automated method.

[0140] Oligonucleotide-based sample labeling In some embodiments, oligonucleotide-based reagents are used to specifically label and image sample components. An example of such a reagent and associated labeling workflow is the CODEX® reagent available from Akoya Biosciences (Menlo Park, CA). Specific embodiments of oligonucleotide-based labeling reagents are described, for example, in U.S. Patent No. 10,370,698, the entire content of which is incorporated herein by reference.

[0141] Oligonucleotide-based reagents can be used to perform multiple imaging cycles. Each imaging cycle can include steps of binding one or more probes to a biological sample, acquiring 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 additional cycles. It should be understood that while oligonucleotide-based reagents are well-suited for performing multiple imaging cycles, other types of reagents, including many of the other types described herein, can also be used to perform such cycles.

[0142] 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 a sample component (target) and an oligonucleotide linked to the binder. Typical sample components include antigens and other protein markers, and specific binders for these markers include, but are not limited to, antibodies and antibody fragments.

[0143] More generally, to target a specific antigen, peptide, protein, or other amino acid-containing species, the binder can include an antibody or antibody fragment. The antibody or antibody fragment can include any one of different types of antibody species, 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 diabodies. The antibody and antibody fragment can be from mouse, rat, rabbit, human, camel, or goat. In some embodiments, the antibody or antibody fragment can be produced against human, mouse, rat, bovine, porcine, ovine, monkey, rabbit, fly, frog, nematode, or woodchuck antigens. In certain embodiments, the antibody or antibody fragment can be produced against antigens of animals, plants, bacteria, fungi, or protists.

[0144] The oligonucleotides linked to the binding agent are specific to each type of binding agent. Thus, for example, in a composition containing three different types of capture agents (each selectively binding to a different sample component), each type of capture agent has the same binding agent linked to the same oligonucleotide. The binding agents among the three types are all different, and the oligonucleotides of each type of capture agent will be different from those of the other types of capture agents.

[0145] In some embodiments, the composition delivered to the biological sample contains only one type of capture agent. However, in certain embodiments, the composition contains multiple types of capture agents. In particular, in a typical sample analysis workflow, it takes a relatively long time to incubate the sample with the capture agent. Thus, by including multiple types of capture agents (corresponding to multiple different targets or components in the sample) in the composition delivered to the sample, many (or all) incubations can be performed simultaneously, significantly improving the efficiency of the analysis.

[0146] In some embodiments, the composition delivered to the sample can contain 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 more) different types of capture agents.

[0147] Each oligonucleotide linked to the capture agent contains a sufficient number of nucleotides such that the probe (discussed below) selectively binds to only one type of capture agent. In certain embodiments, the oligonucleotide contains 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 more) nucleic acids.

[0148] After one or more scavengers have bound to the sample, each type of scavenger is labeled and imaged in one or more labeling, imaging, and label removal cycles. Each such cycle includes three steps. In the first step, the sample is exposed to a composition that includes one or more probes (e.g., by introducing the composition into a chamber formed as described above surrounding the sample). Each composition can include one type of probe, or multiple types of probes. Each type of probe selectively binds to only one of the scavengers in the sample.

[0149] By including multiple types of probes in the composition, multiplexed labeling and imaging of the sample can be performed. Each of the different types of probes in the composition simultaneously binds to different types of scavengers in the sample, significantly shortening the time spent on sample incubation with the probes. In some embodiments, the composition can 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 more) different types of probes.

[0150] Each type of probe includes an oligonucleotide linked to a labeling agent. The oligonucleotide of a single probe type is the same as the labeling agent. However, the oligonucleotide differs depending on the type of probe, similar to the labeling agent. Since each type of probe selectively binds to only one type of scavenger, a particular labeling agent is associated with a particular scavenger (and thus associated with a particular sample component or target). In this way, each type of labeling agent functions as a reporter for a different sample component.

[0151] Each probe oligonucleotide includes a sufficient number of nucleic acids such that specific binding between the pair of scavengers and the probe is achieved. The probe oligonucleotide typically includes 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 more) nucleic acids.

[0152] 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 five or fewer mismatches in perfect complementarity between the nucleic acid sequence of the probe oligonucleotide and at least a portion of the capture agent oligonucleotide.

[0153] A variety of different labeling agents can be linked to the probe oligonucleotide. In some embodiments, for example, the labeling agent includes a fluorescent moiety having different emission center wavelengths, or emission bandwidths, or both.

[0154] The probe can bind to the capture agent by hybridization between the corresponding oligonucleotides. Hybridization can be easily reversed later to remove the probe from the sample. Alternatively, other methods for associating the probe and the capture agent can also be used. In some embodiments, for example, the probe can be bound to a specific capture agent via, for example, ligation or primer extension reactions.

[0155] In the next step of the cycle, the probes bound to the capture agents in the sample are imaged. In particular, a fluorescence image corresponding to the emission from each of the bound probes is obtained. In some embodiments, a fluorescence image containing contributions from substantially only one type of probe can be obtained, for example, by emission filtering. In certain embodiments, the fluorescence image contains non-trivial contributions from multiple probes in the sample, and the individual contributions can be separated using techniques such as spectral unmixing.

[0156] 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 certain embodiments, a residual amount of one or more probes remains in the sample. In the case of a terminal cycle (i.e., the final cycle of the analysis workflow), the probe can be left in the sample to facilitate re-imaging of the sample, for example, after a storage period.

[0157] Generally, to remove the probe from the sample, it is necessary to dehybridize the oligonucleotide of the probe and the corresponding capture agent. Dehybridization can be performed using various techniques. In some embodiments, for example, dehybridization can be carried out by heating the sample to denature the hybridized oligonucleotide. In certain embodiments, the sample can be exposed to one or more dehybridizing agents. Examples of such agents include, but are not limited to, chaotropic reagents such as guanidine hydrochloride, guanidine thiocyanate, and urea.

[0158] Following the removal (or partial removal) of the probe, additional cycles of labeling, imaging, and label removal can be performed to obtain quantitative information about additional components (e.g., biomarkers) in the sample.

[0159] TSA-based signal amplification can also be incorporated into the analysis of sample components via oligonucleotide-based reagents. Methods and reagents for performing TSA amplification using oligonucleotide-based reagents are described, for example, in PCT Patent Application No. PCT / US2020 / 016667 filed on February 4, 2020, the entire content of which is incorporated herein by reference.

[0160] Automated systems for analyzing single and multiple samples The methods described herein can be implemented in a variety of different analysis systems, including systems that perform some or all of the steps in a semi - automatic or fully - automatic manner. An example of such a system 500 is schematically shown in FIG. 5. System 500 includes a memory 502, a labeling 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 related to the controller 508 and can also perform any of the other analysis functions described herein.

[0161] The translation device 510 includes a slide handler 512 that attaches to individual slides using samples within a sealed chamber in order to transport slides between different locations within the system. An example of a sealed chamber and a slide with a sample therein is shown as slide 550 in FIG. 5. The slide handler 512 can be implemented in several ways. In some embodiments, for example, the slide handler 512 is a gripper and includes one or more arms or fingers that apply pressure to the surface of slide 550 to lift and transport slide 550. In certain embodiments, the slide handler 512 includes a member with one or more suction ports that use reduced pressure to lift individual slides 550. In certain embodiments, the slide handler 512 includes one or more members that are inserted under slide 550 to lift the slide. Generally, the slide handler 512 can enable both rotational displacement of slide 550 about three orthogonal axes and translation along the three orthogonal axes.

[0162] The translation device 510 can also include a track or conveyor 513 that carries individual slides 550 or containers of slides between locations within system 500. In some embodiments, the track 513 is a linear track that moves back and forth in one direction between locations. In certain embodiments, the track 513 is a continuous track (e.g., circular, elliptical, or another continuous shape) that circulates between locations within the system.

[0163] During operation, the controller 508 transmits appropriate control signals to the translation device 510 to retrieve one or more slides 550 from the storage unit 502 and, as described above, place one or more slides in the storage unit 502. Further, the controller 508 transmits control signals to the translation device 510 to activate the labeling station and deliver fluids, reagents, and compositions to the chambers of the slide 550 and remove fluids, reagents, compositions (and their components) from the chambers of the slide 550. The labeling station 504 includes one or more reservoirs 518 and a fluid device 514 connected to one or more pumps and / or vacuum sources 519. The fluid device 514 includes one or more fluid conduits 116 (e.g., syringes, tubes) that can be selectively coupled to ports 103 and / or 104 of the chambers of the slide 550. During operation of the system, the controller 508 transmits signals to the translation device 510 to place the slide 550 within the labeling station 504 and transmits signals to the fluid device 514 to couple one or more fluid conduits to one or more ports 103 and / or 104. In this way, fluids, reagents, and compositions are delivered from the reservoir 518 to the chambers of the slide 550 and fluids, reagents, and compositions (and their components) are removed from the chambers, such that the controller 508 can perform any of the staining and labeling operations described herein in an automated manner. Under the control of the controller 508, the labeling station 504 can perform any of the sample preparation steps described herein.

[0164] The 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 can 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 can include one or more different detector types including, but not limited to, CCD detectors and CMOS detectors. During operation of the system 500, to acquire an image of a sample within the chamber of the slide 550, the controller 508 actuates the translation device 510 to position the slide 550 within the imaging station 506. Next, the controller 508 transmits control signals to the components of the imaging station, activating the source 520 to deliver illumination radiation that passes through the beam splitter 522 and the objective lens 524 and impinges on the sample. Light emitted from the sample passes through the objective lens 524, is reflected by the beam splitter 522, and impinges on the image detector 526 that measures an image of the emitted light.

[0165] In FIG. 5, the imaging station is configured to acquire a fluorescent or reflected light image of the sample. However, it should be understood that in some embodiments, the detector 526 can be positioned on the opposite side of the slide 550 from the source 520 to measure a transmitted light image of the sample. In certain embodiments, the imaging station 506 includes multiple detectors for measuring both transmitted light and reflected or emitted light (e.g., fluorescent) images of the sample. Under the control of the controller 508, the imaging station 506 can acquire any of a different types of images corresponding to any of the different types of dyes, probes, and labeling agents described herein.

[0166] As described above, each step of sample analysis consumes a certain amount of time, and the efficiency can be improved by converting multiple slides 550 between multiple locations within the system 500 and performing multiple operations. For example, during the analysis of multiple slides 550, a first slide containing a first sample in the chamber can be placed at the labeling station, and the fluid device can deliver one or more probes to the sample and incubate the sample with the delivered probes. At the same time, a second slide containing a second sample in the chamber can be placed at the imaging station to obtain one or more images of the dye or labeling agent in the sample. A third slide containing a third sample in the chamber can be placed at the labeling station, and the controller 508 can activate one or more pumps and / or vacuum sources to remove the fluid, reagent, and / or composition (and its components) from the chamber of the third slide. A fourth slide containing a fourth sample in the chamber can be placed at the labeling station, and the controller 508 can activate the fluid device 514 to deliver a composition containing one or more primary antibodies to the chamber and incubate the fourth sample with the antibody composition. Other slides 550 can also be processed simultaneously and can undergo any of the operations discussed herein.

[0167] When each slide reaches the end of a set of one or more steps of the preparation or analysis workflow, the slide is transported by the translation device 510 to different locations within the system, such as different stations or different locations within the same station. In this way, the system 500 can analyze multiple samples simultaneously, ensuring that the duty cycle of the components of the system 500 remains relatively high and increasing the overall throughput of the system for multiple samples compared to the simple linear processing of individual samples.

[0168] FIG. 6 shows an example of a controller 508 that can be used with the systems and methods disclosed herein. The controller 508 can include one or more processors 402, a memory 404, a storage device 406, and an interface 408 for interconnecting. The processor 402 can process instructions for execution within the controller, including instructions stored in the memory 404 or the storage device 406. For example, the instructions can direct the processor 402 to perform any of the analysis and control steps disclosed herein.

[0169] The memory 404 can store executable instructions for the processor 402, information regarding system parameters such as excitation and detection wavelengths, and measured spectral image information. The storage device 406 can be a computer-readable medium such as a floppy disk device, a hard disk device, an optical disk device, or a tape device, a flash memory or other similar solid-state memory device, or an array of devices including a storage area network or other configured devices. The storage device 406 can store either instructions executable by the processor 402 described above or other information that can be stored by the memory 404.

[0170] In some embodiments, the controller 508 can include a graphics processing unit for displaying graphic information on an external input / output device such as a display 416 (e.g., using a GUI or text interface). The graphic information can be displayed by a display device (e.g., a CRT (cathode ray tube) or LED (liquid crystal display) monitor) for displaying any of the information such as the measured and calculated spectra and images disclosed herein. The user can provide input to the controller 508 using an input device (e.g., a keyboard, a pointing device, a touch screen, a voice recognition device).

[0171] The user of system 500 can provide the controller 508 with various different types of instructions and information via an input device. The instructions and information can include, for example, information regarding any of the parameters (e.g., staining, labeling, probes, reagents, conditions) related to any of the 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. The controller 508 can use any of these various types of information to execute the methods and functions described herein. It should also be noted that any of these types of information can be stored (e.g., in the storage device 406) and can be called when needed by the controller 508.

[0172] The methods disclosed herein can be implemented by the controller 508 by executing instructions in one or more computer programs executable and / or interpretable by the 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 in a high-level procedural and / or object-oriented programming language and / or in assembly / machine language. For example, the computer program can be stored in the memory 404, the storage 406, and / or a tangible computer-readable medium and can include instructions executable by the processor 402 as described above. As used herein, the term "computer-readable medium" refers to any computer program product, apparatus, and / or device (e.g., magnetic disks, optical disks, memory, programmable logic device (PLD), ASIC, and electronic circuits) used to provide machine instructions and / or data to a programmable processor that receives the machine instructions.

[0173] (Example) (Example: Multiplex IHC Labeling and Histochemical Staining) To demonstrate the effectiveness of the methods described herein, experiments were conducted to analyze FFPE tissue samples via multiplexed IHC labeling and histochemical staining. Samples mounted on microscope slides were heated at a temperature of 55 °C for 30 minutes. Next, the samples were equilibrated at room temperature for approximately 10 minutes and dewaxed. Next, antigen retrieval was performed using tris-ethylenediaminetetraacetic acid (tris-EDTA) and then cooled for 10 minutes. Next, the samples were rinsed three times in deionized water and incubated in acetone for 10 minutes. After removing the samples from the acetone bath and drying them to remove residual acetone, a sealed chamber was formed on the microscope slide using the gasket, window, and two port couplers shown in Figures 1B and 1C.

[0174] Next, the samples were incubated twice for 2 minutes each in a buffer (「S1」) containing EDTA, Dulbecco's phosphate buffered saline (DPBS), BSA, and NaN3. Thereafter, the samples were incubated for 30 minutes in a buffer (「S2」) containing EDTA, DPBS, BSA, NaN3, NaCl, and NaH2PO3. Finally, the samples were incubated for 3 hours in a primary antibody composition containing 2 μg / mL of a primary antibody mixture containing buffer S2, an N-blocker derived from IgG rat, a J-blocker derived from IgG mouse, sheared salmon DNA as an S-blocker, an oligo-based G-blocker, and three different primary antibodies each conjugated to three different oligonucleotides.

[0175] After incubation with the primary antibody, the samples were washed twice with buffer S2 and fixed in a solution of buffer S2 and 1.6% PFA for approximately 4 minutes. Next, the samples were washed three times with DPBS and fixed in methanol for approximately 5 minutes. Next, the samples were washed three times with DPBS, fixed in a solution of DPBS and BS3 for approximately 6 minutes, and then washed three times again with DPBS.

[0176] Finally, the samples were sequentially stained and imaged with DAPI, FITC, and three IHC probes. Each probe contained an oligonucleotide that selectively hybridized to one of the primary antibody oligonucleotides and a distinct fluorescent label.

[0177] Images of each of the dyes and labels applied to the samples are shown in FIGS. 4A-4E. FIG. 4A is an image showing the DAPI distribution within the sample, and FIG. 4B is an image showing the FITC distribution within the sample. FIGS. 4C-4E show the distributions of CD68 (corresponding fluorescent label Cy5), pan-cytokeratin (corresponding fluorescent label AlexaFluor® 750), and E-cadherin (corresponding fluorescent label Atto550). Each of these markers is clearly visible in the corresponding image.

[0178] Other Embodiments Although the present disclosure describes specific implementations, these should not be construed as limitations on the scope of the present disclosure, but rather as descriptions of features in specific embodiments. Features described in the context of separate embodiments may also generally be implemented in combination in a single embodiment. Conversely, the various features described in the context of a single embodiment may also be implemented separately in multiple embodiments or in any suitable sub-combination. Further, features may exist in a particular combination and may initially be claimed as such, but as described above, one or more features from the claimed combination may generally be excised from the combination, and the claimed combination may be directed to a sub-combination or a variation of a sub-combination.

[0179] It will be understood that various modifications to the described embodiments may be made without departing from the spirit and scope of the present disclosure in addition to the embodiments explicitly disclosed herein. Accordingly, other embodiments are within the scope of the following claims.

Description of Reference Numerals

[0180] 101 Second substrate 102 Gasket 103, 103a - 103d, 104, 104a - 104d Ports 105 Inlet manifold 107 Inlet port 110 Outlet manifold 112 Outlet port 113 Opening region 116 Fluid conduit 121 Adhesive layer 123 Adhesive layer 150 First substrate 151 Sample 160 Port coupler 170 First member 172 Second member 174 Opening 175 Elastomer member 176 Magnet 178 Port 180 Fluid conduit 202 Tissue temperature equilibration (RT) 204 Acetone incubation (RT) 206, 306 Drying of tissue (RT) 208, 308 Moisture replenishment of tissue (RT) 210, 310 Tissue blocking (RT) 212, 312 Incubation with primary antibody (RT) 214, 218, 222, 226, 314, 322, 326 Washing (RT) 216, 316 Fixation 1: PFA (RT) 220, 320 Fixation 2: MeOH (4°C) 224, 324 Fixation 3: BS3 (RT) 228, 328 Storage (4°C) 230, 330 One or more labeling, imaging, label removal cycles 302 Deparaffinization (RT) 304 Antigen retrieval (RT) 402 Processor 406 Memory device 500 System 502 Memory Unit 504 Labeling Station 506 Imaging Station 508 Controller 510 Translation Device 512 Slide Handler 513 Track or Conveyor 514 Fluid Device 518 Reservoir 519 Vacuum Source 520 Radiation Source 522 Beam Splitter 524 Objective Lens 526 Image Detector 550 Slide

Claims

Claim 1 A method for analyzing a biological sample, comprising: obtaining a biological sample attached to a first substrate; attaching a second substrate to the first substrate to form a sealed chamber on the first substrate, and disposing the biological sample within the internal volume of the sealed chamber; performing a plurality of imaging cycles, each imaging cycle comprising: (a) binding a probe to the biological sample; (b) obtaining an image of the bound probe in the biological sample; and (c) removing at least a portion of the probe from the biological sample ; and wherein the thickness of the internal volume between the first substrate and the second substrate is 250 micrometers or less.

Images