# US 11,994,452 B2 · Multiplexed tissue imaging > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,994,452 B2 - Document ID: US11994452B2 - Status: Granted - Kind: B2 - Application number: 16/902,215 - Filed: 2020-06-15 - Published: 2020-12-17 - Granted: 2024-05-28 - Earliest claimed: 2019-06-14 - Assignees: Akoya Biosciences, Inc. - Applicants: Akoya Biosciences, Inc. - Inventors: Julia Kennedy-Darling; Peter J. Miller; Peter Harvey - Web page: https://patentgrep.com/patent/US11994452B2 ## CPC - G01N 1/312: {for samples mounted on planar substrates} - G01N 33/5302: {Apparatus specially adapted for immunological test procedures} - G01N 33/5306: {Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding} - G01N 33/552: Glass or silica - G01N 35/00029: {provided with flat sample substrates, e.g. slides (G01N35/028 takes precedence)} - G01N 2035/00039: {Transport arrangements specific to flat sample substrates, e.g. pusher blade} - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract 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. ## Claims ### Claim 1 A method for analyzing a biological sample, the method comprising: obtaining a biological sample mounted on a first substrate; affixing a second substrate to the first substrate to form an enclosed chamber on the first substrate with the biological sample positioned within an interior volume of the enclosed chamber, wherein the second substrate comprises a first port formed by a first aperture extending through a thickness of the second substrate and a second port formed by a second aperture extending through the thickness of the second substrate; introducing a composition into the interior volume through the first port, wherein the composition comprises multiple different types of capture agents, each of the different types of capture agents comprising a different type of binding agent that selectively binds to a different sample component and an oligonucleotide linked to the binding agent that is specific to each different type of binding agent, wherein each of the different types of capture agents is bound to the biological sample by introducing the different types of capture agents at the same time into the interior volume of the enclosed chamber; and performing multiple imaging cycles, wherein each imaging cycle comprises: (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 a thickness of the interior volume between the first and second substrates is 250 micrometers or less. ### Claim 2 (depends on 1) The method of claim 1, wherein the first substrate is formed of a substantially transparent material. ### Claim 3 (depends on 1) The method of claim 1, wherein the second substrate comprises a window. ### Claim 4 (depends on 1) The method of claim 1, further comprising positioning a gasket between the first and second substrates, wherein the gasket is formed of an adhesive material. ### Claim 5 (depends on 1) The method of claim 1, further comprising, prior to affixing the second substrate to the first substrate: incubating the biological sample in acetone; removing residual acetone from the biological sample; and affixing the second substrate to the first substrate following the removal of residual acetone. ### Claim 6 (depends on 1) The method of claim 1, further comprising, prior to performing the multiple imaging cycles, applying a stain to the biological sample. ### Claim 7 (depends on 1) The method of claim 1, wherein the interior volume is 0.20 cm3 or less. ### Claim 8 (depends on 1) The method of claim 1, wherein during each imaging cycle, the probe is bound to the biological sample by introducing a composition comprising the probe through the first port, and removing components of the composition through the second port. ### Claim 9 (depends on 1) The method of claim 1, wherein binding the probe to the biological sample comprises binding one or more different types of probes to the biological sample, wherein each different type of probe comprises a different type of oligonucleotide linked to a labeling agent that is specific to each different type of oligonucleotide. ### Claim 10 (depends on 9) The method of claim 9, wherein the one or more different types of probes are bound to the biological sample at the same time by introducing a composition comprising the one or more different types of probes into the interior volume of the enclosed chamber, and wherein the one or more different types of probes bind to corresponding different types of capture agents in the biological sample. ### Claim 11 A method for analyzing a biological sample, the method comprising: obtaining a biological sample mounted on a first substrate; affixing a second substrate to the first substrate to form an enclosed chamber on the first substrate with the biological sample positioned within an interior volume of the enclosed chamber, wherein the second substrate comprises a first port formed by a first aperture extending through a thickness of the second substrate and a second port formed by a second aperture extending through the thickness of the second substrate; introducing a composition comprising multiple different types of capture agents through the first port, each of the different types of capture agents comprising a different type of binding agent that selectively binds to a different sample component and an oligonucleotide linked to the binding agent that is specific to each different type of binding agent; and performing multiple imaging cycles, wherein each imaging cycle comprises: (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 and second substrates are substantially transparent; and wherein an imaging window formed by the first and second substrates has a field of view of at least 500 mm2. ### Claim 12 (depends on 11) The method of claim 11, wherein the first substrate comprises a microscope slide. ### Claim 13 (depends on 11) The method of claim 11, wherein the second substrate comprises a window. ### Claim 14 (depends on 11) The method of claim 11, further comprising positioning a gasket between the first and second substrates, wherein the gasket is formed of an adhesive material. ### Claim 15 (depends on 11) The method of claim 11, further comprising, prior to affixing the second substrate to the first substrate: incubating the biological sample in acetone; removing residual acetone from the biological sample; and affixing the second substrate to the first substrate following the removal of residual acetone. ### Claim 16 (depends on 11) The method of claim 11, further comprising, prior to performing the multiple imaging cycles, applying a stain to the biological sample. ### Claim 17 (depends on 11) The method of claim 11, wherein a minimum distance between the first and second ports measured in a plane of the second substrate is 20 mm or more. ### Claim 18 (depends on 11) The method of claim 11, wherein during each imaging cycle, the probe is bound to the biological sample by introducing a composition comprising the probe through the first port, and removing components of the composition through the second port. ### Claim 19 (depends on 11) The method of claim 11, wherein each of the different types of capture agents is bound to the biological sample by introducing the composition comprising each of the different types of capture agents at the same time into the interior volume of the enclosed chamber. ### Claim 20 (depends on 16) The method of claim 16, wherein binding the probe to the biological sample comprises binding one or more different types of probes to the biological sample, wherein each different type of probe comprises a different type of oligonucleotide linked to a labeling agent that is specific to each different type of oligonucleotide. ### Claim 21 (depends on 16) The method of claim 16, further comprising, prior to introducing the composition into the interior volume of the enclosed chamber, connecting a port coupler to at least one of the first and second ports. ### Claim 22 A method for analyzing a biological sample, the method comprising: obtaining a biological sample mounted on a first substrate; affixing a second substrate to the first substrate to form an enclosed chamber on the first substrate with the biological sample positioned within an interior volume of the enclosed chamber, wherein the second substrate comprises a first port formed by a first aperture extending through a thickness of the second substrate and a second port formed by a second aperture extending through the thickness of the second substrate, and wherein compositions are introduced into the interior volume through the first port; prior to introducing a composition into the interior volume of the enclosed chamber, connecting a port coupler to at least one of the first and second ports; and performing multiple imaging cycles, wherein each imaging cycle comprises: (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 and second substrates are substantially transparent. ### Claim 23 (depends on 22) The method of claim 22, wherein the first substrate is formed of a substantially transparent material. ### Claim 24 (depends on 22) The method of claim 22, wherein the second substrate comprises a window. ### Claim 25 (depends on 22) The method of claim 22, further comprising positioning a gasket between the first and second substrates, wherein the gasket is formed of an adhesive material. ### Claim 26 (depends on 22) The method of claim 22, further comprising, prior to affixing the second substrate to the first substrate: incubating the biological sample in acetone; removing residual acetone from the biological sample; and affixing the second substrate to the first substrate following the removal of residual acetone. ### Claim 27 (depends on 22) The method of claim 22, further comprising, prior to performing the multiple imaging cycles, applying a stain to the biological sample. ### Claim 28 (depends on 22) The method of claim 22, wherein during each imaging cycle, the probe is bound to the biological sample by introducing a composition comprising the probe through the first port, and removing components of the composition through the second port. ### Claim 29 (depends on 22) The method of claim 22, further comprising, prior to performing the multiple imaging cycles, binding a capture agent to the biological sample by introducing a composition comprising the capture agent through the first port. ### Claim 30 (depends on 29) The method of claim 29, wherein the capture agent comprises a binding agent that selectively binds to a sample component, and an oligonucleotide that is linked to the binding agent. ### Claim 31 (depends on 30) The method of claim 30, wherein the composition comprises multiple different types of capture agents, each of the different types of capture agents comprising a different type of binding agent that selectively binds to a different sample component, and an oligonucleotide that is specific to each different type of binding agent. ### Claim 32 (depends on 31) The method of claim 31, wherein each of the different types of capture agents is bound to the biological sample by introducing the composition comprising each of the different types of capture agents at the same time into the interior volume of the enclosed chamber. ### Claim 33 (depends on 22) The method of claim 22, wherein binding the probe to the biological sample comprises binding one or more different types of probes to the biological sample, wherein each different type of probe comprises a different type of oligonucleotide linked to a labeling agent that is specific to each different type of oligonucleotide. ### Claim 34 (depends on 33) The method of claim 33, wherein the one or more different types of probes are bound to the biological sample at the same time by introducing a composition comprising the one or more different types of probes into the interior volume of the enclosed chamber, and wherein the one or more different types of probes bind to corresponding different types of capture agents in the biological sample. ### Claim 35 (depends on 22) The method of claim 22, wherein the interior volume is 0.20 cm3 or less.