WO2024145224A1

WO2024145224A1 - Compositions, methods, and systems for high resolution spatial analysis

Info

Publication number: WO2024145224A1
Application number: PCT/US2023/085717
Authority: WO
Inventors: Hanyoup Kim; Tobias Daniel Wheeler; Nabil Mikhaiel
Original assignee: 10X Genomics Inc
Current assignee: 10X Genomics Inc
Priority date: 2022-12-29
Filing date: 2023-12-22
Publication date: 2024-07-04
Anticipated expiration: 2025-06-29
Also published as: WO2024145224A9

Abstract

Systems, apparatuses, and methods of preparing a substrate for use in spatial analysis of a biological sample are provided. The method can include providing a first substrate including a first surface. The method can also include providiung a second substrate including a first surface. The second substrate can include an array area comprising an array of capture probes. At least one capture probe of the array of capture probes can include a capture domain. The method can further include forming at least one gap-defining element on the first surface of the second substrate. The method can also include applying the second substrate to the first surface of the first substrate. The method can further include forming a reagent dispensing element on the first substrate surrounding the array area of the second substrate.

Description

COMPOSITIONS, METHODS, AND SYSTEMS FOR HIGH RESOLUTION SPATIAL

ANALYSIS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63/435,860, filed on December 29, 2022. The disclosure of the above-referenced application is herein expressly incorporated by reference it its entirety.

BACKGROUND

[0002] Cells within a tissue of a subject have differences in cell morphology and/or function due to varied analyte levels (e.g., gene and/or protein expression) within the different cells. The specific position of a cell within a tissue (e.g., the cell’s position relative to neighboring cells or the cell’s position relative to the tissue microenvironment) can affect, e.g., the cell’s morphology, differentiation, fate, viability, proliferation, behavior, and signaling and cross-talk with other cells in the tissue.

[0003] Spatial heterogeneity has been previously studied using techniques that only provide data for a small handful of analytes in the context of an intact tissue or a portion of a tissue, or provide a lot of analyte data for single cells, but fail to provide information regarding the position of the single cell in a parent biological sample (e.g., tissue sample).

[0004] Analytes from a biological sample can be captured onto an array while preserving spatial context of the analytes using fluid reagents. The captured analytes can be used to generate a sequence data that can be mapped to an image of the biological sample. There exists a need for methods and systems that enable management of fluid reagents to perform high resolution spatial analysis of biological samples. Acquiring images of biological samples at sufficient detail to enable high resolution spatial analyses and visualizations of analyte distributions at one or more locations of the biological sample is limited by existing sample handling apparatuses and image acquisition methods used therein.

SUMMARY

[0005] In one aspect, a substrate for spatial analysis of a biological sample is provided. In one embodiment, the substrate can include a first substrate comprising a first surface and a second substrate positioned atop the first surface of the first substrate. The second substrate can include an array area on a first surface of the second substrate. The array area can include an array of capture probes. At least one capture probe of the array of capture probes can include a capture domain. The substrate can also include at least one gap-defining clement extending vertically from the first surface of the second substrate. The substrate can further include a reagent dispensing element on the first surface of the first substrate, the reagent dispensing element surrounding the array area of the second substrate and the at least one gap-defining element. [0006] In another embodiment, the capture probe can further include a spatial barcode. In another embodiment, the capture probe can further include a spatial barcode, a unique molecular identifier (UMI), one or more functional domains including a primer binding site, or a combination thereof. For example, in some embodiments, the capture probe includes a capture domain (e.g., a random sequence, a fixed sequence such as a poly(T)), a spatial barcode, a unique molecular identifier (UMI), and a primer binding site. In another embodiment, the at least one gap-defining element can surround the array area. In another embodiment, the at least one gapdefining element can have a height of about 50 microns or less. In another embodiment, the at least one gap-defining element can have a height of less than 2 microns, or has a height of 1.5 microns, or has a height of about 1 micron.

[0007] hi another embodiment, a second gap-defining element can extend vertically from the first surface of the first substrate. In another embodiment, the substrate can further include a third substrate opposite the first surface of the first substrate. The at least one gap-defining element can be configured to maintain a gap height between the first substrate and the third substrate. In another embodiment, the third substrate can include a biological sample (e.g., a tissue sample such as a tissue section). In another embodiment, the first substrate and the third substrate can form a fluidically sealed chamber there between when the reagent dispensing element is compressed between the first substrate and the third substrate. In another embodiment, the array area of the second substrate, the at least one gap-defining element, and the third substrate can form a fluidically sealed chamber.

[0008] In another embodiment, the at least one gap-defining element can include a first material and the reagent dispensing area can include at least one second material different than the first material. In another embodiment, the at least one first material can include a positive photoresist material. In another embodiment, the at least one first material can include a negative photoresist material. In another embodiment, the reagent dispensing element can include a plurality of second materials arranges in a plurality of layers. In another embodiment, the at least one second material can include a hydrophobic material and/or a material including reduced friction properties. In another embodiment, the hydrophobic material can include a polymer, silicone, glass, or polydimethylsiloxane.

[0009] In another embodiment, the reagent dispensing element can be configured to control an amount of a fluid dispersed between the first substrate and the third substrate comprising a biological sample, suppress a flow of a fluid present between the first substrate and the third substrate comprising a biological sample as the first substrate and the third substrate are brought into contact with the reagent dispensing element, reduce bubble formation in a fluid present between the first substrate and the third substrate comprising a biological sample as the first substrate and the third substrate are brought into contact with the reagent dispending element, or identify a reagent dispensing area configured on the second substrate. In another embodiment, the substrate can include a plurality of gap-defining elements, each gap-defining element positioned at a respective corner of the second substrate. In another embodiment, the first substrate can include a glass slide.

[0010] In another aspect, a substrate for spatial analysis of a biological sample is provided and can include a first substrate having a first surface. The substrate can also include a second substrate coupled to the first surface of the first substrate. The second substrate can include an array area on a first surface of the second substrate, wherein the array area comprises an array of features. A first feature of the array can include one or more first capture probes. The one or more first capture probes can include a first capture domain and a first spatial barcode that identifies a location of the first feature, and a second feature of the array can include one or more second capture probes. The one or more second capture probes can include a second capture domain and a second spatial barcode that identifies a location of the second feature. The first feature or the second feature can have a diameter, or a maximum distance between adjacent first features or second features, of 500 nm to 2 pm, I pm to 3 pm, 1 pm to 5 pm, or 1 pm to 10 pm. The substrate can also include at least one gap-defining element extending vertically from the first surface of the second substrate. The substrate can further include a reagent dispensing element on the first surface of the first substrate, the reagent dispensing element surrounding the array area on the first surface of the second substrate and having a height of 750 microns or more. In another embodiment, the at least one gap-defining element has a height of less than 50 microns, less than 40 microns, less than 30 microns, less than 20 microns, less than 10 microns, less than 5 microns, or less than 2 microns, or has a height of about 1 .5 microns, or has a height of about 1 micron.

[0011] In another aspect, a method for spatial analysis of a biological sample is provided and can include providing a sample handling apparatus holding a first substrate, a second substrate, and a reagent medium. The first substrate can include a biological sample mounted thereon. The biological sample can include an analyte, the second substrate can include a reagent dispensing element and a third substrate can include an array comprising a plurality of capture probes and at least one gap-defining element. At least one capture probe of the plurality of capture probes can include a capture domain. The method can also include aligning the first substrate with the second substrate. The aligning can assemble a chamber comprising the first substrate, the second substrate, the biological sample, and the reagent dispensing element. The reagent dispensing element can be positioned to at least partially surround or fully surround an area on the first substrate on which the biological sample is disposed and/or the array disposed on the third substrate. The area of the first substrate, the reagent dispensing element, and the second substrate can at least partially enclose a volume comprising the biological sample and the at least one gap-defining element can maintain a gap height between the first substrate and the second substrate as the reagent dispensing element is compressed between the first substrate and the second substrate once the chamber is assembled. The method can further include releasing the analyte from the biological sample when the first substrate and second substrates are aligned. The releasing can cause the analyte to migrate from the biological sample to the array where the analyte can be captured by a capture probe.

[0012] In another embodiment, the migrated analyte can hybridize to the at least one capture probe of the plurality of capture probes. In another embodiment, the at least one capture probe of the plurality of capture probes can further include a spatial barcode. In another embodiment, the analyte can migrate to the array via passive diffusion. In another embodiment, the analyte can include a nucleic acid such as DNA, RNA, or a ligated RTL probe. In another embodiment, the reagent medium can include a permeabilization agent. In another embodiment, the reagent medium can include a protease, a detergent, or an RNAse.

[0013] In another embodiment, the sample handling apparatus can include a first member that receives the first substrate, a second member that receives the second substrate, and an adjustment mechanism configured to align the first substrate and the second substrate to assemble the chamber comprising the first substrate, the second substrate, the biological sample, and the reagent dispensing clement. In another embodiment, the biological sample and the capture probe can be contacted with the reagent medium when the first substrate is aligned with the second substrate.

[0014] hi another embodiment, the method can include generating a barcoded polynucleotide comprising a sequence of the analyte or a sequence of a complement of the analyte, and the spatial barcode of the capture probe or a complement of the spatial barcode of the capture probe. In another embodiment, the method can include determining the sequence of the analyte or the complement thereof included in the barcoded polynucleotide. In another embodiment, the method can include determining a location of the analyte within the biological sample based on the determined sequences of the analyte and spatial barcode included in the barcoded polynucleotide. In some embodiments, generating the barcoded polynucleotide includes extending the at least one capture probe using the analyte as a template, thereby generating an extended capture probe, and/or extending the analyte using the capture probe as a template, thereby generating an extended analyte. In some embodiments, generating the barcoded polynucleotide further includes amplifying the extended capture probe and/or extended analyte. [0015] In another aspect a method of preparing a substrate for use in spatial analysis of a biological sample is provided and, in one embodiment, can include providing a first substrate comprising a first surface. The method can also include providing a second substrate comprising a first surface. The second substrate can include an array area comprising an array of capture probes. At least one capture probe of the array of capture probes can include a capture domain. In other embodiments, all the capture probes include a capture domain. The capture probe can further include a spatial barcode, a unique molecular identifier (UMI), one or more functional domains (e.g., a primer binding site), or a combination thereof. The method can further include applying the second substrate onto the first surface of the first substrate. The method can also include forming a reagent dispensing element on the first substrate surrounding the array area of the second substrate.

[0016] In another embodiment, providing the second substrate can include providing an array base substrate comprising a first surface. Providing the second substrate can also include forming a plurality of arrays on the first surface of the array base substrate, each array of the plurality of arrays comprising a plurality of array elements. Providing the second substrate can further include forming at least one gap-defining element on the first surface of the array base substrate for each array of the plurality of arrays. The at least one gap-defining clement can extending vertically from the first surface of the array base substrate. Providing the second substrate can also include dicing the array base substrate to form one or more second substrates. Each second substrate of the one or more second substrates can include at least one array comprising the plurality of array elements, and the at least one gap-defining element.

[0017] In another embodiment, the at least one gap-defining element can surround the array area. In another embodiment, the at least one gap-defining element can extend vertically from the first surface of the first substrate. In another embodiment, the at least one gap-defining element can be formed from a first material and the reagent dispensing element can be formed from at least one second material.

[0018] In another embodiment, the first material can be a positive photoresist material and forming the at least one gap-defining element on the first substrate or the second substrate can include exposing the positive photoresist material to ultraviolet light at a location on the first substrate or the second substrate corresponding to the at least one gap-defining element, and applying a developer solution to remove the positive photoresist material from the location on the first substrate or the second substrate corresponding to the at least one gap-defining element. [0019] In another embodiment, the first material can be a negative photoresist material and forming the at least one gap-defining element on the first substrate or the second substrate can include exposing the negative photoresist material to ultraviolet light at a location on the first substrate or the second substrate corresponding to a location of the at least one gap-defining element, and applying a developer solution to remove the negative photoresist material from the location on the first substrate or the second substrate corresponding to the at least one gapdefining element.

[0020] In another embodiment, forming the reagent dispensing element can include forming a plurality of layers using a plurality of second materials. In another embodiment, the at least one second material can include a hydrophobic material. In another embodiment, the hydrophobic material can include a polymer, silicone, glass, or polydimethylsiloxane.

[0021] In another embodiment, the method of preparing a substrate for use in spatial analysis of a biological sample can include providing a third substrate opposite the first surface of the first substrate. The at least one gap-defining element can be configured to maintain a gap height between the first substrate and the third substrate. In another embodiment, the third substrate can include a biological sample. In another embodiment, the biological sample can include a fresh and/or frozen tissue section or a fixed tissue section. In another embodiment, the first substrate and the third substrate can form a fluidically sealed chamber there between when the reagent dispensing element is compressed between the first substrate and the third substrate. In another embodiment, forming the at least one gap-defining element can include forming the at least one gap defining element to have a height of about 50 microns or less. In another embodiment, the at least one gap-defining element can have a height of less than 2 microns, or has a height of about 1.5 microns, or has a height of about 1 micron.

[0022] In another embodiment, the reagent dispensing element can be configured to control an amount of a fluid dispersed between the first substrate and the third substrate, suppress a flow of a fluid present between the first substrate and the third substrate as the first substrate and the third substrate are brought into contact with the reagent dispensing element, reduce bubble formation in a fluid present between the first substrate and the third substrate as the first substrate and the third second substrate are brought into contact with the reagent dispensing element, or identify a reagent dispensing area configured on the second substrate. In another embodiment, the method of preparing a substrate for use in spatial analysis of a biological sample can include forming a plurality of gap-defining elements. Each gap-defining element can be formed at a respective comer of the second substrate.

[0023] All publications, patents, patent applications, and information available on the internet and mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, patent application, or item of information was specifically and individually indicated to be incorporated by reference. To the extent publications, patents, patent applications, and items of information incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

[0024] Where values are described in terms of ranges, it should be understood that the description includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated. [0025] The term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but docs not necessarily refer to every item in the collection, unless expressly stated otherwise, or unless the context of the usage clearly indicates otherwise. [0026] Use of ordinal terms such as “first”, “second”, “third”, etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. Similarly, the use of these terms in the specification does not by itself connote any required priority, precedence, or order.

[0027] In the descriptions above and in the claims, phrases such as “at least one of’ or “one or more of’ may occur followed by a conjunctive list of elements or features. The term “and/or” may also occur in a list of two or more elements or features. Unless otherwise implicitly or explicitly contradicted by the context in which it used, such a phrase is intended to mean any of the listed elements or features individually or any of the recited elements or features in combination with any of the other recited elements or features. For example, the phrases “at least one of A and B;” “one or more of A and B;” and “A and/or B” are each intended to mean “A alone, B alone, or A and B together.” A similar interpretation is also intended for lists including three or more items. For example, the phrases “at least one of A, B, and C;” “one or more of A, B, and C;” and “A, B, and/or C” are each intended to mean “A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together.” Use of the term “based on,” above and in the claims is intended to mean, “based at least in part on,” such that an unrecited feature or element is also permissible.

[0028] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. If the degree of approximation is not otherwise clear from the context, “about” means either within plus or minus 10% of the provided value, or rounded to the nearest significant figure, in all cases inclusive of the provided value. In some embodiments, the term “about” indicates the designated value ± up to 10%, up to ± 5%, or up to ± 1%.

[0029] Various embodiments of the features of this disclosure are described herein. However, it should be understood that such embodiments are provided merely by way of example, and numerous variations, changes, and substitutions can occur to those skilled in the art without departing from the scope of this disclosure. It should also be understood that various alternatives to the specific embodiments described herein are also within the scope of this disclosure.

DESCRIPTION OF DRAWINGS

[0030] The following drawings illustrate certain embodiments of the features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner. Like reference symbols in the drawings indicate like elements.

[0031] FIG. 1 is a schematic diagram depicting an exemplary permeabilization solution interaction between a first substrate (e.g., tissue slide) and a second substrate (e.g., gene expression slide) in a sandwich configuration in accordance with some example implementations.

[0032] FIG. 2A is a perspective view of an example sample handling apparatus in a closed position in accordance with some example implementations.

[0033] FIG. 2B is a perspective view of the example sample handling apparatus in an open position in accordance with some example implementations.

[0034] FIG. 3A shows an exemplary sandwich configuration in accordance with some example implementations.

[0035] FIG. 3B shows a fully formed sandwich creating a chamber formed from the one or more spacers, the first substrate, and the second substrate in accordance with some example implementations .

[0036] FIG. 3C depicts a top view of the configuration of FIG. 3B.

[0037] FIG. 4 depicts an example configuration for venting or removing bubbles from the chamber in accordance with some example implementations.

[0038] FIG. 5A is a side view of the angled closure workflow in accordance with some example implementations. [0039] FIG. 5B is a top view of the angled closure workflow in accordance with some example implementations .

[0040] FIGS. 6A-6E show an example workflow for an angled sandwich assembly in accordance with some example implementations.

[0041] FIGS. 7A-7C depict a side view and a top view of an angled closure workflow for sandwiching a first substrate having a tissue sample and a second substrate in accordance with some example implementations.

[0042] FIG. 8 is a diagram of an example system software architecture in accordance with some example implementations.

[0043] FIG. 9A depicts a top view of a substrate including a reagent dispensing element configured for use in the sample handling apparatus described herein in accordance with some example implementations.

[0044] FIG. 9B depicts a cross-sectional view of the substrate of FIG. 9A.

[0045] FIG. 9C depicts a cross-sectional view of the substrate of FIG. 9B in an initial sandwich configuration with a second substrate configured for use in the sample handling apparatus described herein in accordance with some example implementations.

[0046] FIGS. 10A-10C depict a sandwiching process using the substrate of FIGS. 9A-9C configured for use in the sample handling apparatus described herein in accordance with some example implementations.

[0047] FIG. 11 A depicts an embodiment of a gap-defining element included in the substrate of FIGS. 9A-9C in accordance with some example implementations.

[0048] FIG. 1 IB depicts another embodiment of a gap-defining element included in the substrate of FIGS. 9A-9C in accordance with some example implementations.

[0049] FIG. 12A depicts an embodiment of a reagent dispensing element included in the substrate of FIGS. 9A-9C in accordance with some example implementations.

[0050] FIG. 12B depicts an embodiment of a reagent dispensing element included in the substrate of FIGS. 9A-9C in accordance with some example implementations.

[0051] FIG. 12C depicts an embodiment of a reagent dispensing element included in the substrate of FIGS. 9A-9C in accordance with some example implementations. [0052] FIG. 13 is a diagram illustrating a method of forming a gap-defining element configured for use with the sample handling apparatus described herein using a positive photoresist material in accordance with some example implementations.

[0053] FIG. 14 is a diagram illustrating a method of forming a gap-defining element configured for use with the sample handling apparatus described herein using a negative photoresist material in accordance with some example implementations.

[0054] FIG. 15 is a process flow diagram illustrating an example process for forming a plurality of array substrates including gap-defining elements in accordance with example implementations.

[0055] FIG. 16 is a process flow diagram illustrating an example process for forming an array substrate according to some implementations of the current subject matter.

[0056] FIG. 17A depicts a top-down view of an experimental workflow for forming a substrate including a reagent dispensing element and a spacer configured for use in the sample handling apparatus described herein in accordance with some example implementations.

[0057] FIG. 17B depicts a side-view of the experimental workflow of FIG. 17A.

[0058] FIGS. 18A-18C depicts an embodiment of a sandwiching workflow used in the experimental workflow of FIGS. 17A-17B.

[0059] FIGS. 19A-19B depict a Gaussian fit used to detect a center spot on the substrate formed in the experimental workflow of FIGS. 17A-17B.

[0060] FIGS. 19C depicts a plot of flow data over time based on the Gaussian fit method used in FIGS. 19A-19B.

[0061] FIGS. 20A-20F depict time-series image data of a sample of a substrate formed via the experimental workflow of FIGS. 17A-17B.

[0062] FIGS. 21A-21C depict plots of experimental results showing the flow data for samples of substrates formed via the experimental workflow of FIGS. 17A-17B.

DETAILED DESCRIPTION

Introduction

[0063] This disclosure describes apparatus, systems, methods, and compositions for spatial analysis of biological samples. This section describes certain general terminology, analytes, sample types, and preparative steps that are referred to in later sections of the disclosure. For example, the terms and phrases: spatial analysis, barcode, nucleic acid, nucleotide, probe, target, oligonucleotide, polynucleotide, subject, genome, adaptor, adapter, tag, hybridizing, hybridize, annealing, anneal, primer, primer extension, proximity ligation, nucleic acid extension, polymerase chain reaction (PCR) amplification, antibody, affinity group, label, detectable label, optical label, template switching oligonucleotide, splint oligonucleotide, analytes, biological samples, general spatial array-based analytical methodology, spatial analysis methods, immunohistochemistry and immunofluorescence, capture probes, substrates, arrays, analyte capture, partitioning, analysis of captured analytes, quality control, multiplexing, and/or the like are described in more detail in PCT Patent Application Publication No. W02020/123320, the entire contents of which are incorporated herein by reference.

[0064] Tissues and cells can be obtained from any source. For example, tissues and cells can be obtained from single-cell or multicellular organisms (e.g., a mammal). The relationship between cells and their relative locations within a tissue sample may aid understanding disease pathology. Spatialomic (e.g., spatial transcriptomic) technology may allow scientists to measure all the gene activity in a tissue sample and map where the activity is occurring. This technology and embodiments described herein may lead to new discoveries that may prove instrumental in helping scientists gain a better understanding of biological processes and disease.

[0065] Tissues and cells obtained from a mammal, e.g., a human, often have varied analyte levels (e.g., gene and/or protein expression) which can result in differences in cell morphology and/or function. The position of a cell or a subset of cells (e.g., neighboring cells and/or nonneighboring cells) within a tissue can affect, e.g., the cell’s fate, behavior, morphology, and signaling and crosstalk with other cells in the tissue. Information regarding the differences in analyte levels (gene and/or protein expression) within different cells in a tissue of a mammal can also help physicians select or administer a treatment that will be effective and can allow researchers to identify and elucidate differences in cell morphology and/or cell function in the single-cell or multicellular organisms (e.g., a mammal) based on the detected differences in analyte levels within different cells in the tissue. Differences in analyte levels within different cells in a tissue of a mammal can also provide information on how tissues (e.g., healthy and diseased tissues) function and/or develop. Differences in analyte levels within different cells in a tissue of a mammal can also provide information of different mechanisms of disease pathogenesis in a tissue and mechanism of action of a therapeutic treatment within a tissue. [0066] The spatial analysis methodologies herein provide for the detection of differences in an analyte level (c.g., gene and/or protein expression) within different cells in a tissue of a mammal or within a single cell from a mammal. For example, spatial analysis methodologies can be used to detect the differences in analyte levels (e.g., gene and/or protein expression) within different cells in histological slide samples, the data from which can be reassembled to generate a three- dimensional map of analyte levels (e.g., gene and/or protein expression) of a tissue sample obtained from a mammal, e.g., with a degree of spatial resolution (e.g., single-cell resolution). [0067] Spatial heterogeneity in developing systems has typically been studied via RNA hybridization, immunohistochemistry, fluorescent reporters, or purification or induction of predefined subpopulations and subsequent genomic profiling (e.g., RNA-seq). Such approaches, however, rely on a relatively small set of pre-defined markers, therefore introducing selection bias that limits discovery. These prior approaches also rely on a priori knowledge. RNA assays traditionally relied on staining for a limited number of RNA species. In contrast, single-cell RNA-sequencing allows for deep profiling of cellular gene expression (including non-coding RNA), but the established methods separate cells from their native spatial context.

[0068] Spatial analysis methodologies described herein provide a vast amount of analyte level and/or expression data for a variety of multiple analytes within a sample at high spatial resolution, e.g., while retaining the native spatial context.

[0069] The binding of an analyte to a capture probe can be detected using a number of different methods, e.g., nucleic acid sequencing, fluorophore detection, nucleic acid amplification, detection of nucleic acid ligation, and/or detection of nucleic acid cleavage products. In some examples, the detection is used to associate a specific spatial barcode with a specific analyte produced by and/or present in a cell (e.g., a mammalian cell).

[0070] Capture probes can be, e.g., attached to a surface, e.g., a solid array, a bead, or a coverslip. In some examples, capture probes are not attached to a surface. In some examples, capture probes can be encapsulated within, embedded within, or layered on a surface of a permeable composition (e.g., any of the substrates described herein).

[0071] Non-limiting aspects of spatial analysis methodologies are described in

WO 2011/127099, WO 2014/210233, WO 2014/210225, WO 2016/162309, WO 2018/091676, WO 2012/140224, WO 2014/060483, U.S. Patent No. 10,002,316, U.S. Patent No. 9,727,810, U.S. Patent Application Publication No. 2017/0016053, Rodriques et al., Science 363(6434): 1463-1467, 2019; WO 2018/045186, Lee et al., Nat. Protoc. 10(3):442-458, 2015;

WO 2016/007839, WO 2018/045181, WO 2014/163886, Trejo ct al., PLoS ONE

14(2) :e0212031, 2019, U.S. Patent Application Publication No. 2018/0245142, Chen et al., Science 348(6233):aaa6090, 2015, Gao et al., BMC Biol. 15:50, 2017, WO 2017/144338, WO 2018/107054, WO 2017/222453, WO 2019/068880, WO 2011/094669, U.S. Patent No. 7,709,198, U.S. Patent No. 8,604,182, U.S. Patent No. 8,951,726, U.S. Patent No. 9,783,841, U.S. Patent No. 10,041,949, WO 2016/057552, WO 2017/147483, WO 2018/022809, WO 2016/166128, WO 2017/027367, WO 2017/027368, WO 2018/136856, WO 2019/075091, U.S. Patent No. 10,059,990, WO 2018/057999, WO 2015/161173, and Gupta et al., Nature Biotechnol. 36:1197-1202, 2018, the entire contents of which are incorporated herein by reference and can be used herein in any combination. Further non-limiting aspects of spatial analysis methodologies are described herein.

[0072] Embodiments described herein may map the spatial patterns of analytes in complex tissue samples (e.g., on tissue slides) with slides (e.g., gene expression slides), by utilizing analyte (e.g., mRNA transcript) capture and spatial barcoding technology for library preparation. A tissue (e.g., fresh-frozen, formalin fixed paraffin-embedded (FFPE), or the like) may be sectioned and placed in proximity to a slide with an array of barcoded spots, each containing capture oligonucleotides with spatial barcodes unique to that spot. Once tissue sections are fixed, stained, and permeabilized, they release analytes (e.g., mRNA) which binds to capture oligos from a proximal location on the tissue. A barcoding reaction ((e.g., an extension reaction such as, e.g., reverse transcription) may occur, generating a cDNA library that incorporates the spatial barcodes and preserves spatial information. Barcoded cDNA libraries may be further processed to prepare sequencing libraries. Sequence information from the libraries may be mapped back to a specific spot on a capture area of the barcoded spots. This sequence data may be subsequently layered over a high-resolution microscope image of the tissue section, making it possible to visualize the sequence data within the morphology of the tissue in a spatially resolved manner. As described herein, the term “barcoded spots” can be considered equivalent and synonymous with the terms “array elements”, and “array features” and can be used interchangeably, except where noted otherwise.

[0073] In one embodiment of a workflow for preparing a biological sample on a slide, a glass slide, can be provided. The workflow may further include placing tissue sections on the glass slide. Placing tissue sections on the glass slide may include placing the tissue anywhere on the glass slide including placing the tissue on or in relation to a fiducial disposed on the glass slide. The fiducial may include any marking to aid in placement of the tissue on the slide and/or aid in the alignment of the tissue slide relative to the gene expression slide. The workflow can further include staining the tissue with hematoxylin and/or eosin stain or another staining agent or method. The workflow can further include imaging the tissue on the slide using brightfield (to image the sample using hematoxylin and/or eosin stain) or another imaging technique. The imaging may include high-resolution imaging on a user imaging system. The imaging may allow the user to confirm the relevant pathology and/or identify any target areas for analysis.

[0074] Embodiments described herein relating to preparing the biological sample on the slide may beneficially allow a user to confirm pathology or relevant regions on a tissue section, to confirm selection of best or undamaged tissue sections for analysis, to improve array-tissue alignment by allowing placement anywhere on the pathology slide. Further, workflows for preparing the biological sample on the slide may empower user or scientists to choose what to sequence (e.g., what tissue section(s) to sequence).

[0075] FIG. 1 is a schematic diagram depicting an exemplary sandwiching process 100 between a first substrate comprising a biological sample (e.g., a tissue section 102 on a slide 103) and a second substrate comprising an array, e.g., a slide 104 that is populated with features containing capture probes 106. In some embodiments, the array is spatially barcoded, e.g., the capture probes 106 are spatially barcoded. While FIG. 1 depicts slide 104 as having barcoded capture probes 106, it is to be understood that capture probes 106 need not be barcoded. During the exemplary sandwiching process, the first substrate is aligned with the second substrate, such that at least a portion of the biological sample is aligned with at least a portion of the array (e.g., aligned in a sandwich configuration). As shown, the array slide 104 is in a superior position to the tissue slide 103. In some embodiments, the tissue slide 103 may be positioned superior to the slide 104. When a permeabilization solution 105 is applied to a gap 107 between the tissue slide 103 and the array slide 104, the permeabilization solution 105 creates a permeabilization buffer which permeabilizes or digests the sample 102 and the analytes (e.g., mRNA transcripts) 108 of the tissue sample 102 may release, actively or passively migrate (e.g., diffuse) across the gap 107 toward the capture probes 106, and bind (e.g., hybridize) on the capture probes 106. [0076] After the analytes (e.g., transcripts) 108 bind on the capture probes 106, an extension reaction may occur, thereby generating a spatially barcoded library. For example, in the case of mRNA transcripts, reverse transcription may be used to generate a cDNA library associated with a particular spatial barcode. Barcoded cDNA libraries may be mapped back to a specific spot on a capture area of the capture probes 106. This gene expression data may be subsequently layered over a high-resolution microscope image of the tissue section, making it possible to visualize the expression of any mRNA, or combination of mRNAs, within the morphology of the tissue in a spatially resolved manner. In some embodiments, the extension reaction can be performed separately from the sample handling apparatus described herein that is configured to perform the exemplary sandwiching process 100. The sandwich configuration of the sample 102, the pathology slide 103 and the array slide 104 may provide advantages over other methods of spatial analysis and/or analyte capture. For example, the sandwich configuration may reduce a burden of users to develop in house tissue sectioning and/or tissue mounting expertise. Further, the sandwich configuration may decouple sample preparation/tissue imaging from the barcoded array (e.g., spatially barcoded capture probes 106) and enable selection of a particular region of interest of analysis (e.g., for a tissue section larger than the barcoded array). The sandwich configuration also beneficially enables spatial analysis without having to place a tissue section 102 directly on the array slide (e.g., slide 104).

[0077] The sandwich configuration described herein further provides the beneficial ability to quality check or select specific sections of tissue prior to committing additional time and resources to the analysis workflow. This can be advantageous to reduce costs and risk or mistakes or issues that can arise during sample preparation. Additionally, the sandwich configuration an enable the ability to select which area of a sample to sequence when a sample section is larger than an array. Another benefit of using the sandwich configuration described herein is the ability to separate fiducial imaging and high-resolution sample imaging. This can enable the separation of expertise required to perform histology workflows and molecular biology workflows and can further enable the assay and the sample to be moved between different laboratories. Additionally, the sandwich configuration described herein can provide great flexibility and more options in sample preparation conditions since there are no oligos on the sample substrate or slide. This can reduce the likelihood a sample may fall off the substrate and can reduce the likelihood that oligos are damaged due to high temperatures or interactions with other reagents during sample preparation. The sandwich configuration described herein can also improve the sensitivity and spatial resolution by vertically confining target molecules within the diffusion distance.

Fluid Delivery Systems and Methods

[0078] Analytes within a biological sample are generally released through disruption (e.g., permeabilization, digestion, etc.) of the biological sample or may be released without disruption. Various methods of permeabilizing (e.g., any of the permeabilization reagents and/or conditions described herein) a biological sample are described herein, including for example including the use of various detergents, buffers, proteases, and/or nucleases for different periods of time and at various temperatures. Additionally, various methods of delivering fluids (e.g., a buffer, a permeabilization solution) to a biological sample are described herein including the use of a substrate holder (e.g., sandwich assembly, sandwich configuration, as described herein).

[0079] Provided herein are methods for delivering a fluid to a biological sample disposed on an area of a first substrate and an array disposed on a second substrate.

[0080] In some embodiments and with reference to FIG. 1, the sandwich configuration described herein between a tissue sample slide (e.g., slide 103) and a gene expression slide (e.g., the array slide 104 with barcoded capture probes 106) may involve the addition of a liquid reagent (e.g., permeabilization solution 105 or other target molecule release and capture solution) to fill a gap (e.g., gap 107). It may be desirable that the liquid reagent be free from air bubbles between the slides to facilitate transfer of target molecules (e.g., nucleic acids) with spatial information. Additionally, air bubbles present between the slides may obscure at least a portion of an image capture of a desired region of interest. Accordingly, it may be desirable to ensure or encourage suppression and/or elimination of air bubbles between the two slides during a permeabilization step.

[0081] In some aspects, it may be possible to reduce or eliminate bubble formation between the slides using a variety of filling methods and/or closing methods.

[0082] Workflows described herein may include contacting a drop of the liquid reagent disposed on a first substrate (e.g., the first slide 103) or a second substrate (e.g., the second slide 104) with at least a portion of a first substrate (e.g., the first slide 103) or second substrate (e.g., the second slide 104), respectively. In some embodiments, the contacting comprises bringing the two substrates into proximity such that the sample on the first substrate is aligned with the barcode array of capture probes on the second substrate.

[0083] In some embodiments, the drop includes permeabilization reagents (e.g., any of the permeabilization reagents described herein). In some embodiments, the rate of permeabilization of the biological sample is modulated by delivering the permeabilization reagents (e.g., a fluid containing permeabilization reagents) at various temperatures.

[0084] In some embodiments, the permeabilization reagents are dried permeabilization reagents. In some embodiments, the dried permeabilization reagents are disposed on a substrate (e.g., the first substrate, the second substrate). In some embodiments, delivering the fluid (e.g., by any of the fluid delivery methods described herein) solubilizes the dried permeabilization reagents. In some embodiments, solubilizing the permeabilization reagents results in permeabilization of the biological sample. In some embodiments, delivering the fluid to solubilize dried reagents is delivered via an aperture in a gasket. In some embodiments, delivering the fluid to solubilize dried reagents is delivered through a via-hole. In some embodiments, the fluid solubilizing dried reagents includes the use of a syringe. In some embodiments, the fluid solubilizing dried reagents includes the capillary flow.

[0085] In the example sandwich maker workflows described herein, a liquid reagent (e.g., the permeabilization solution 105) may fill a gap (e.g., the gap 107) between a tissue slide (e.g., slide 103) and a capture slide (e.g., slide 104 with barcoded capture probes 106) to warrant or enable transfer of target molecules with spatial information. Described herein are examples of filling methods that may suppress bubble formation and suppress undesirable flow of transcripts and/or target molecules or analytes. Robust fluidics in the sandwich making described herein may preserve spatial information by reducing or preventing deflection of molecules as they move from the tissue slide to the capture slide.

[0086] FIG. 3A shows an exemplary sandwiching process 300 where a first substrate (e.g., slide 303), including a biological sample 302 (e.g., a tissue section), and a second substrate (e.g., slide 304 including spatially barcoded capture probes 306) are brought into proximity with one another. As shown in FIG. 3A a liquid reagent drop (e.g., permeabilization solution 305) is located on the second substrate in proximity to the capture probes 306 and in between the biological sample 302 and the second substrate (e.g., slide 304). While FIG. 3A shows the liquid reagent drop 305 located on slide 304, it is also understood that drop 305 may be located on one or more spacers 310. The permeabilization solution 305 may release analytes that can be captured by the capture probes 306 of the array. As further shown, one or more spacers 310 may be positioned between the first substrate (e.g., pathology slide 303) and the second substrate (e.g., slide 304). The one or more spacers 310 may be configured to maintain a separation distance between the first substrate and the second substrate, e.g., when brought into a fully formed sandwich configuration, e.g., as shown in FIG. 3B. While the one or more spacers 310 is shown as disposed on the second substrate, the spacer may additionally or alternatively be disposed on the first substrate.

[0087] FIG. 3B shows a fully formed sandwich configuration creating a chamber 350 formed from the one or more spacers 310, the first substrate (e.g., the pathology slide 303), and the second substrate (e.g., the slide 304) in accordance with some example implementations. In the example of FIG. 3B, the liquid reagent (e.g., the permeabilization solution 305) fills the volume of the chamber 350 and may create a permeabilization buffer that allows analytes 308 (e.g., mRNA transcripts and/or other molecules) to diffuse from the biological sample 302 toward the capture probes 306 of the slide 304. In some aspects, flow of the permeabilization buffer may deflect the analytes 308 from the biological sample 302 and may affect diffusive transfer of analytes 308 for spatial analysis. A partially or fully sealed chamber 350 resulting from the one or more spacers 310, the first substrate, and the second substrate may reduce or prevent flow from undesirable convective movement of analytes 308 over the diffusive transfer from the biological sample 302 to the capture probes 306.

[0088] FIG. 3C depicts a top view of the configuration 325 of FIG. 3B. As shown, the one or more spacers 310 may fully enclose and surround the biological sample 302 and form the chamber 350 when sandwiched between the first substrate and the second substrate. The right hand side of FIG. 3C depicts an example of reduced convection during by capturing images of the sample 302 at the start of the sandwich and at the end of the sandwich. Half of such images may be stitched together to pronounce the dominant diffusion and suppressed convection during sandwiching.

[0089] The spacers 310 can provide a separation distance between the first and second substrates to advantageously enable high resolution spatial analysis of analytes captured on an array (e.g., the array on the second substrate as described herein). The high-resolution spatial analysis of captured analytes can be performed using spacers that are dimensioned to produce higher spatial frequencies of acquired image data. As a result, high resolution image data can be acquired and used to provide spatial analyses of analyte distributions present within a biological sample. In some aspects, the spacer configurations provided herein allow for high resolution capture of analytes from a biological sample (c.g., a biological sample mounted on a first substrate) to an array of features described herein (e.g., a second substrate comprising the array as disclosed herein).

[0090] During sandwiching and permeabilization, the permeabilization reagent digest the tissue and target molecules diffuse across the sandwich gap from the tissue to the array and bind on the barcoded oligos on the array. High resolution spatial imaging and analysis can be achieved when the molecule diffusive broadening is limited. This diffusive broadening is directly proportional to the sandwich gap height, thus the smaller the gap height, the higher spatial resolution can be achieved. More specifically, referring to the sandwich configuration in FIG. 1, smaller gap heights, e.g., less than 12.5 pm can advantageously reduce diffusive broadening to achieve high resolution spatial analysis. For example, for a high-resolution substrate with a capture spot size of 5 pm, a sandwich gap height equal or smaller than 5 um advantageously provides targeted high resolution spatial analysis information with accuracy and sufficient quality. Thin spacers configured for use with the substrates of the sample handling apparatus are provided herein for advantageously creating the desired sandwich gap height.

[0091] In some embodiments, the one or more spacers 310 is configured to maintain a separation distance between first and second substrates that is less than 12.5 microns. In some embodiments, the one or more spacers 310 is configured to maintain a separation distance between first and second substrates that is between about 2 microns and 10 microns, measured in a direction orthogonal to the surface of first substrate that supports the sample. In some embodiments, the one or more spacers 310 is configured to maintain a separation distance between first and second substrates that is between about 0.5 microns and about 10 microns, between about 0.5 and about 5 microns, between about 1 micron and about 5 microns, between about 2.5 microns and about 5 microns. In some instances, the one or more spacers 310 is configured to maintain a separation distance between first and second substrates that is about 5 microns or less. In some instances, the one or more spacers 310 is configured to maintain a separation distance between first and second substrates that is about 0.5 microns. In some instances, the one or more spacers 310 is configured to maintain a separation distance between first and second substrates that is about 1 microns. In some instances, the one or more spacers 310 is configured to maintain a separation distance between first and second substrates that is about 1.5 microns. In some instances, the one or more spacers 310 is configured to maintain a separation distance between first and second substrates that is about 2 microns. In some instances, the one or more spacers 310 is configured to maintain a separation distance between first and second substrates that is less than 2 microns. In some instances, the one or more spacers 310 is configured to maintain a separation distance between first and second substrates that is about 2.5 microns. In some instances, the one or more spacers 310 is configured to maintain a separation distance between first and second substrates that is about 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 microns. In some embodiments, the separation distance is measured in a direction orthogonal to a surface of the first substrate that supports the biological sample.

[0092] In some embodiments, the one or more spacers 310 have a height that is less than 12.5 microns. In some embodiments, the one or more spacers 310 have a height that is between about 2 microns and 10 microns, measured in a direction orthogonal to the surface of first substrate that supports the sample. In some embodiments, the one or more spacers 310 have a height that is between about 0.5 microns and about 10 microns, between about 0.5 and about 5 microns, between about 1 micron and about 5 microns, or between about 2.5 microns and about 5 microns. In some instances, the one or more spacers 310 have a height that is about 5 microns or less. In some instances, the one or more spacers 310 have a height that is about 0.5 microns. In some instances, the one or more spacers 310 have a height that is about 1 microns. In some instances, the one or more spacers 310 have a height that is about 1.5 microns. In some instances, the one or more spacers 310 have a height that is about 2 microns. In some instances, the one or more spacers 310 have a height that is about 2.5 microns. In some instances, the one or more spacers 310 have a height that is about 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 microns.

[0093] FIG. 4 depicts an example configuration 400 for venting or removing bubbles from the chamber 350 in accordance with some example implementations. FIG. 4 depicts a top view of the chamber 350 where the square portion includes the capture probes 306, the circular portion includes the biological sample 302, and the rectangular’ portion includes a hydrophobic area 420. The hydrophobic area 420 may include a hydrophobic pattern that does not wet and is disposed in a portion of the chamber 350 that is located away from an area of interest (e.g., an area where the biological sample 302 and the capture probes 306 overlap). The hydrophobic area 420 may be configured to remove or reduce bubbles (e.g., bubbles 615) from the chamber 350 during the permeabilization step. [0094] In some aspects, any combination of bubble venting or bubble removing features may be applied to the chamber, the first substrate, and/or the second substrate. For example, air permeable spacers (e.g., spacers 310) may be configured to vent out trapped bubbles. Further, bubble venting holes disposed on the first substrate, the second substrate, and/or a spacer may be placed at strategic locations to vent bubbles. In some aspects, a sonication or vibration device may be configured to generate vibration on the first substrate and/or the second substrate during closing of the sandwich to reduce the chance of a bubble sticking to a surface of the first substrate or the second substrate. Additionally, it may be possible to increase a humidity of the chamber during sandwich closing to facilitate the filling process of the permeabilization solution or liquid reagent. Further, it may be possible to generate a vacuum in the chamber during closing to reduce or eliminate the chance of bubble trapping.

[0095] FIG. 5A is a side view of the angled closure workflow 500 in accordance with some example implementations. FIG. 5B is a top view of the angled closure workflow 500 in accordance with some example implementations. As shown at step 405, the drop 401 is positioned to the side of the slide 402.

[0096] At step 410, the drop side of the angled slide 406 contacts the drop 401 first. The contact of the slide 406 with the drop 401 may form a linear or low curvature flow front that fills uniformly with the slides closed.

[0097] At step 415, the slide 406 is further lowered toward the slide 402 (or the slide 402 is raised up toward the slide 406) and the dropped side of the slide 406 may contact and may urge the liquid reagent toward the side opposite the dropped side and creating a linear or low curvature flow front that may prevent or reduce bubble trapping between the slides.

[0098] At step 425, the drop 401 of liquid reagent fills the gap (e.g., the gap 107) between the slide 406 and the slide 402. The linear flow front of the liquid reagent may form by squeezing the drop 405 volume along the contact side of the slide 402 and/or the slide 406. Additionally, capillary flow may also contribute to filling the gap area.

[0099] FIGS. 6A-6E show an example workflow 600 for an angled sandwich assembly in accordance with some example implementations. As shown in FIG. 6 A, the base 604 may be positioned tilted at an angle. The slide 612 may be disposed flat on the base 604 and at the same angle. The angle may be determined such that a drop (e.g., drop 605) placed on the surface of the slide 612 will not fall off the surface (e.g., due to gravity). The angle may be determined by a gravitational force versus any surface force to move the drop away from the off the slide 612.

[0100] FIG. 6B depicts the slide 606 and the slide 612 being sandwich together as the slide 606 and the slide 612 move toward each other and the slide 606 contacts the drop 605. In some aspects, the slides 606 and 612 may be parallel or at an angle relative to each other during the sandwiching. In some embodiments the angle of the slides may be achieved via a sample handling apparatus described herein (e.g., the sample handling apparatus 200 or the like).

[0101] FIG. 6C depicts one or more air bubbles 615 trapped within the drop 605 during the sandwiching of the slides 606 and 612.

[0102] As shown in FIG. 6D, the one or more air bubbles 615 may be less dense than the liquid reagent drop 605 and the one or more air bubbles 615 may migrate up in a superior direction due to buoyancy. In some aspects, as the one or more air bubbles 615 reach the top (e.g., uppermost part of the drop 605), the bubbles may release or otherwise be removed from the drop 605.

[0103] FIG. 6E depicts the base 604, the slide 606, and the slide 612 straightened along an axis and the one or more bubbles 615 removed from the drop 605 or removed from a region of interest between the slides 606 and 612.

[0104] In some aspects, the angled closure of FIGS. 5A-5B, 6A-6E, and 7A-7C may occur in response to detecting a bubble (e.g., bubble 615) within the drop 605. Additionally or alternatively, the angled closures described herein may occur during each sandwiching of the slides (e.g., the slides 606 and 612). A sensor may be configured to detect a bubble in the liquid reagent drop 605 responsive to a slide (e.g., the slide 606) or a tissue sample (e.g., tissue sample 602) contacting at least a portion of the drop 605.

[0105] FIGS. 7A-7C depict a side view and a top view of an angled closure workflow 700, with reference to FIGS. 3A-3C, for sandwiching a first substrate (e.g., slide 303) having a tissue sample 302 and a second substrate (e.g., slide 304 having capture probes 306) in accordance with some example implementations.

[0106] FIG. 7A depicts the first substrate (e.g., the slide 303 including sample 302) angled over (superior to) the second substrate (e.g., slide 304). As shown, a drop of the permeabilization solution 305 is located on top of the spacer 310 toward the right-hand side of the side view in FIG. 7A. [0107] FIG. 7B shows that as the first substrate lowers, or as the second substrate rises, the dropped side of the first substrate (c.g., a side of the slide 303 angled inferior to the opposite side) may contact the drop of the permeabilization solution 305. The dropped side of the first substrate may urge the permeabilization solution 305 toward the opposite direction. For example, in the side view of FIG. 7B the permeabilization solution 305 may be urged from right to left as the sandwich is formed.

[0108] FIG. 7C depicts a full closure of the sandwich between the first substrate and the second substrate with the spacer 310 contacting both the first substrate and the second substrate and maintaining a separation distance between the two. As shown in the top view of FIGS. 7B-7C, the spacer 310 fully encloses and surrounds the tissue sample 302 and the capture probes 306, and the spacer 310 forms the sides of chamber 350 which holds a volume of the permeabilization solution 305.

[0109] In some aspects, the alignment of the tissue sample 302 with the capture probes 306 shown in FIGS. 7A-7C may be performed by an alignment mechanism of a sample handling apparatus described herein (e.g., sample handling apparatus 200, or the like).

Systems and Methods for Sample Analysis

[0110] The methods described above for analyzing biological samples, such as the sandwich configuration described above, can be implemented using a variety of hardware components. In this section, examples of such components are described. However, it should be understood that in general, the various steps and techniques discussed herein can be performed using a variety of different devices and system components, not all of which are expressly set forth.

[0111] In some embodiments, a sample handling apparatus, also referred to as a sample holder, includes a first member that receives a first substrate on which a sample may be positioned. The first member may include a first retaining mechanism configured to retain the first substrate in a fixed position along an axis and disposed in a first plane. In some embodiments, the sample handling apparatus also includes a second member that receives a second substrate. The second member may include a second retaining mechanism configured to retain the second substrate disposed in a second plane. The second substrate may include an array, e.g., a barcoded array (e.g., array of spatially barcoded capture probes), as described above. In some embodiments, the sample handling apparatus also includes an adjustment mechanism configured to move the second member. The adjustment mechanism may be coupled to the second member and include a linear actuator configured to move the second member along a z axis orthogonal to the second plane. In some aspects, the adjustment mechanism may be alternatively or additionally coupled to the first member. In some embodiments, an adjustment mechanism coupled to the first member comprises a linear actuator configured to move the first member along a z axis orthogonal to the first plane.

[0112] FIG. 2A is a perspective view of an example sample handling apparatus 200 in a closed position in accordance with some example implementations. As shown, the sample handling apparatus 200 includes a first member 204, a second member 210, an image capture device 220, a first substrate 206, a hinge 215, and a mirror 216. The hinge 215 may be configured to allow the first member 204 to be positioned in an open or closed configuration by opening and/or closing the first member 204 in a clamshell manner along the hinge 215.

[0113] FIG. 2B is a perspective view of the example sample handling apparatus 200 in an open position in accordance with some example implementations. As shown, the sample handling apparatus 200 includes one or more first retaining mechanisms 208 configured to retain one or more first substrates 206. In the example of FIG. 2B, the first member 204 is configured to retain two first substrates 206, however the first member 204 may be configured to retain more or fewer first substrates 206.

[0114] In some aspects, when the sample handling apparatus 200 is in an open position (e.g., as shown in FIG. 2B), the first substrate 206 and/or the second substrate 212 may be loaded and positioned within the sample handling apparatus 200 such as within the first member 204 and the second member 210, respectively. As noted, the hinge 215 may allow the first member 204 to close over the second member 210 and form a sandwich configuration (e.g., the sandwich configuration shown in FIG. 1).

[0115] In some aspects, after the first member 204 closes over the second member 210, an adjustment mechanism (not shown) of the sample handling apparatus 200 may actuate the first member 204 and/or the second member 210 to form the sandwich configuration for the permeabilization step (e.g., bringing the first substrate 206 and the second substrate 212 closer to each other and within a threshold distance for the sandwich configuration). The adjustment mechanism may be configured to control a speed, an angle, or the like of the sandwich configuration. [0116] In some embodiments, the tissue sample (e.g., sample 302) may be aligned within the first member 204 (e.g., via the first retaining mechanism 208) prior to closing the first member 204 such that a desired region of interest of the sample 302 is aligned with the bar-coded array of the gene expression slide (e.g., the slide 304) , e.g., when the first and second substrates are aligned in the sandwich configuration. Such alignment may be accomplished manually (e.g., by a user) or automatically (e.g., via an automated alignment mechanism). After or before alignment, spacers may be applied to the first substrate 206 and/or the second substrate 212 to maintain a minimum spacing between the first substrate 206 and the second substrate 212 during sandwiching. In some aspects, the permeabilization solution (e.g., permeabilization solution 305) may be applied to the first substrate 206 and/or the second substrate 212. The first member 204 may then close over the second member 210 and form the sandwich configuration. Analytes 308 (e.g., mRNA transcripts) may be captured by the capture probes 306 and may be processed for spatial analysis.

[0117] In some embodiments, during the permeabilization step, the image capture device 220 may capture images of the overlap area between the tissue 302 and the capture probes 306. If more than one first substrates 206 and/or second substrates 212 are present within the sample handling apparatus 200, the image capture device 220 may be configured to capture one or more images of one or more overlap areas.

System and Methods for Alignment of a Sample and an Array

[0118] Spatial analysis workflows described herein generally involve contacting a sample with an array of features or elements (e.g., barcoded spots). With such workflows, aligning the sample with the array is an important step in performing spatialomic (e.g., spatial transcriptomic) assays. The ability to efficiently generate robust experimental data for a given sample can depend greatly on the alignment of the sample and the array. Traditional techniques require samples to be placed directly onto the array. This approach can require skilled personnel and additional experimental time to prepare a section of the sample and to mount the section of the sample directly on the array. Misalignment of the sample and the array can result in wasted resources, extended sample preparation time, and inefficient use of samples, which may be limited in quantity.

[0119] The systems, methods, and computer readable mediums described herein can enable efficient and precise alignment of samples and arrays, thus facilitating the spatialomic (e.g., spatial transcriptomic) imaging and analysis workflows or assays described herein. Samples, such as portions of tissue, can be placed on a first substrate. The first substrate can include a slide onto which a user can place a sample of the tissue. An array can be formed on a second substrate. The second substrate can include a slide and the array can be formed on the second substrate.

The use of separate substrates for the sample and the array can beneficially allow user to perform the spatialomic (e.g., spatial transcriptomic) assays described herein without requiring the sample to be placed onto an array substrate. The sample holder and methods of use described herein can improve the ease by which users provide samples for spatialomic (e.g., spatial transcriptomic) analysis. For example, the systems and methods described herein alleviate users from possessing advanced sample or tissue sectioning or mounting expertise. Additional benefits of utilizing separate substrates for samples and arrays can include improved sample preparation and sample imaging times, greater ability to perform region of interest (ROI) selection, and more efficient use of samples and array substrates. The systems, methods, and computer readable mediums described herein can further enable users to select the best sections of a sample to commit to sequencing workflows. Some tissue samples or portions of the tissue samples can be damaged during mounting. For examples, the tissue samples or portions of the tissue samples can be folded over on themselves. The systems, methods, and computer readable mediums described herein can further enable users to confirm relevant pathology and/or biology prior to committing to sequencing workflows.

[0120] The sample substrate and the array substrate, and thus, the sample and the array, can be aligned using the instrument and processes described herein. The alignment techniques and methods described herein can generate more accurate spatialomic (e.g., spatial transcriptomic) assay results due to the improved alignment of samples with an array.

[0121] In some embodiments, a workflow described herein comprises contacting a sample disposed on an area of a first substrate with at least one feature array of a second substrate. In some embodiments, the contacting comprises bringing the two substrates into proximity such that the sample on the first substrate may be aligned with the barcoded array on the second substrate. In some instances, the contacting is achieved by arranging the first substrate and the second substrate in a sandwich assembly. In some embodiments, the workflow comprises a prior step of mounting the sample onto the first substrate. [0122] Alignment of the sample on the first substrate with the array on the second substrate may be achieved manually or automatically (c.g., via a motorized alignment). In some aspects, manual alignment may be done with minimal optical or mechanical assistance and may result in limited precision when aligning a desired region of interest for the sample and the barcoded array. Additionally, adjustments to alignment done manually may be time-consuming due to the relatively small time requirements during the permeabilization step.

[0123] It may be desirable to perform real-time alignment of a tissue slide (e.g., the pathology slide 303) with an array slide (e.g., the slide 304 with barcoded capture probes 306). In some implementations, such real-time alignment may be achieved via motorized stages and actuators of a sample handling apparatus described herein (e.g., the sample handling apparatus 200, or the like).

Exemplary Computer Systems and Architectures

[0124] In some aspects, the example sample handling apparatuses described herein may implement software to provide some of the functions of the sample handling apparatus. For example, software may be used to control aspects of image processing, substrate alignment, substrate temperature control, instrument safety, or the like.

[0125] FIG. 8 is a diagram of an example system architecture 2600 in accordance with some example implementations described herein. For example, the system architecture 2600 can be configured to perform one or more of workflows and processes described herein. As shown in FIG. 8, the sample handling apparatus 200 may include an input/output control board 2605, a camera control 2610, and a network interface 2620. As shown, the input/output control board 2605, the camera control 2610, and the network interface 2615 may be connected via a controller area network (CAN) bus. The input/output control board 2605 may be configured to control aspects or components of the sample handling apparatus 200. For example, the input/output control board 2605 can include a controller and may be configured to control a pump, a fan, a motor of a linear actuator, one or more sensors, a heater, a TEC, or the like. The camera control 2610 may be configured to control aspects or components of a camera (e.g., the image capture device 220). For example, the camera control 2610 may control a focus, a zoom, a position of the camera, an image capture, or the like.

[0126] The sample handling apparatus 200 also includes a processor 2620, a memory 2625, an input device 2630, and a display 2635. The processor 2620 can be configured to execute computer- readable instructions stored the memory 2625 to perform the workflows and processes described herein. The processor 2620 can also execute computer-readable instructions stored in the memory 2625, which cause the processor 2620 to control operations of the sample handling apparatus 200 via the I/O control board 2605 and/or the image capture device 220 via the camera control 2610. In this way, the processor 2620 can control an operation of the sample handling apparatus 200 to align a sample with an array. For example, the processor 2620 can execute instructions to cause either of the first retaining mechanism or the second retaining mechanism to translate within the sample handling apparatus 200 so as to adjust their respective locations and to cause a sample area of a first substrate to be aligned with an array area of a second substrate.

[0127] The input device 2630 can include a mouse, a stylus, a touchpad, a joy stick, or the like configured to receive user inputs from a user. For example, a user can use the input device 2630 to provide an input indicating a sample area indicator for a first substrate. The display 2635 can include a graphical user interface 2640.

[0128] The network interface 2615 may be configured to provide wired or wireless connectivity with (e.g., via Ethernet, Wi-Fi, or the like) a network 2645, such as the Internet, a local area network, a wide area network, a virtual private network, or the like. The network 2645 may be connected to one or more distributed computing resources, such as a cloud computing environment, a software as a service (SaaS) pipeline 2650, and/or a support portal 2655. The SaaS pipeline 2650 may be configured to aid or control automated image alignment or other alignment. The support portal 2655 may be configured to send images/videos/logs to the support portal and for issues to debug. The sample handling apparatus 400 can also be communicatively coupled via the network 2645 to a second computing device 2660 located remotely from the location of the sample handling apparatus 200.

Reagent Flow Management and Substrate Spacing Features

[0129] FIG. 9A is a diagram illustrating a reagent dispensing element 2810 and a gap-defining element 2830 configured on a substrate for use with the sample handling apparatus described herein in accordance with some example implementations. As shown in FIG. 9A, a reagent dispensing element 2810 can be formed on or coupled to a substrate 2800. The substrate 2800 can be considered a "‘first” substrate and can be configured to include one or more analysis regions 2805, such as region 2805A and 2805B. Each analysis region 2805 can include a reagent dispensing element 2810 upon which a reagent medium 2815 can be located or provided. The reagent dispensing element 2810 can include one or more layers that can be formed from materials that vary in their elasticity. For example, the reagent dispensing clement 2810 can be applied to the first substrate 2800 and can include a compressible material. In some embodiments, the reagent dispensing element 2810 can include a first layer formed from a rigid base material affixed to the substrate 2800 and a second layer formed from a compressible material affixed to the first layer including the rigid base material. The reagent dispensing element 2810 can be configured to aid dispensing of the reagent medium 2815 onto the array 2825 and to suppress flow of the reagent medium 2815 in a controlled manner.

[0130] A “second” substrate 2820 can be applied to the first substrate 2800. The second substrate 2820 can include an array area including an array 2825 including a plurality of capture probes as descried herein. The second substrate 2820 can also include a gap-defining element 2830 arranged with respect to the array 2825. The gap-defining element 2830 can be configured from a rigid material and can be further configured to create, maintain or provide a gap-height between adjacent substrates in a sandwich configuration for high resolution capture of analytes released during permeabilization (e.g., of a biological sample) onto the array 2825.

[0131] One of skill in the ail will recognize the terms “first substrate”, “second substrate”, and/or “third substrate” can be relative terms describing one, two, or three respective substrates and are not intended to limit features associated with respective substrates to any one particular substrate except where otherwise noted. In some embodiments, the substrate 2800 can be a “first substrate”, the substrate 2820 can be a “second substrate”, and the substrate 2835 can be a “third substrate”. In some embodiments, the substrate 2820 can be a “first substrate” and the substrate 2800 or 2835 can be a “second substrate”. In some embodiments, the substrate 2820 can be a “third substrate” and the substrate 2800 and 2835 can be the “first substrate” and the “second substrate”, respectively. A variety of indexed labeling (e.g., “first”, “second”, “third”) can be envisioned for describing the various substrates herein. In some embodiments, the gap-defining element 2830 can be formed on or coupled to the substrate 2820. In some embodiments, the gap-defining element 2830 can be formed on or coupled to the substrate 2800. In some embodiments, the reagent dispensing element 2810 can be formed on or coupled to the substrate 2800. In some embodiments, the substrates 2800, 2820, and/or 2835 can include a glass material, a plastic material, or a silicon material. In some embodiments, the reagent dispensing element 2810 can include a glass material, a plastic material, or a silicon material. In some embodiments, the gap- defining element 2830 can include a rigid material. In some embodiments, the gap-defining element 2830 can include a photo-patterned photoresist material such as an epoxy-based polymer, a polyimide-based polymer, an electron beam cross-linked polymer, or a polymer formed via deepultraviolet light photolithography. In some embodiments, the gap-defining element 2830 can include an adhesive applied polymer or a thermally bonded polymer. In some embodiments, the gap-defining elements 2830 can include transparent or opaque materials such that the substrate 2820 can be imaged through the gap-defining element 2830.

[0132] As shown in FIG. 9B, the gap-defining element 2830 can include a height Hl corresponding to a predetermined gap height to be maintained when the substrates 2800 and 2820 are brought into contact with substrate 2835 in a sandwich configuration. For example, the gapdefining element 2830 can provide a controlled gap height Hl between the substrates 2820 and 2835. In some embodiments, the gap-defining element 2830 can have a height Hl less than 10 microns, although other heights can be envisioned. In some embodiments, the gap-defining element 2830 has a height Hl that is between about 2 microns and 10 microns, measured in a direction orthogonal to the surface of substrate 2820 (e.g., the surface on which the array 2825 is formed, or the surface of substrate 2820 facing substrate 2835 shown in FIG. 9C). In some embodiments, the gap-defining element 2830 has a height that is between about 0.5 microns and about 10 microns, between about 0.5 and about 5 microns, between about 1 micron and about 5 microns, or between about 2.5 microns and about 5 microns. In some instances, the gap-defining element 2830 has a height that is about 5 microns or less. In some embodiments, the gap-defining element 2830 has a height that is about 0.5 microns. In some embodiments, the gap-defining element 2830 has a height that is about 1 micron. In some embodiments, the gap-defining element 2830 has a height that is about 1.5 microns. In some embodiments, the gap-defining element 2830 has a height that is about 2 microns. In some instances, the gap-defining element 2830 has a height that is about 2.5 microns. In some instances, the gap-defining element 2830 has a height that is about 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 microns. In some embodiments, the gap-defining element 2830 can have a height that is about 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45- 50, or 50-55 microns. In some embodiments, the gap-defining element can have a width of 250 microns. The width of the gap-defining element 2830 can be measured transversely to its height Hl. [0133] The reagent dispensing element 2810 can include a height H2 and the substrate 2820 can include a height H3. In some embodiments, the height H2 of the reagent dispensing clement 2810 can be greater than the height H3 of the substrate 2820. In this way, material of the reagent dispensing element 2810 can be compressed to form a sealed chamber around the substrate 2820 (and thus array 2825) and in between the substrate 2800 and 2835. In some embodiments, the height H2 of the reagent dispensing element 2810 can be configured with respect to the height Hl of the gap-defining element 2830 and/or the height H3 of the substrate 2820. In some embodiments, the reagent dispensing element 2810 can have a height H2 of about 600-900 microns, for example about 700 microns when the reagent dispensing element 2810 includes an uncompressible material. In some embodiments, the reagent dispensing element 2810 can include a compressible material and the height H2 can be about 800 microns. In some embodiments, the substrate 2820 can have a height H3 of about 700-800 microns, for example about 750 microns. Broadly, the substrate 2820 can include a variety of non-limiting heights H3. The height H3 of the substrate 2820 can, in part, be determined by a thickness of the wafer that the die is diced from. Wafers can have thicknesses that are about 275, 375, 525, 625, 675, 725, 775, or 925 microns.

[0134] The reagent dispensing element 2810 can include a height H2 that is slightly larger or smaller than the height H3 of the substrate 2820 depending on the material of the reagent dispensing element 2810. For example, the height H2 of the reagent dispensing element 2810 can be smaller (e.g., about 50 microns smaller) than the height H3 of the substrate 2820 when the reagent dispensing element 2810 includes an incompressible material or can be larger (e.g., about 50 microns larger) than the height H3 of the substrate 2820 when the reagent dispensing element 2810 includes a compressible material. In some embodiments, when the reagent dispensing element 2810 includes a compressible material, height H2 can be larger than the sum of height H3 of the substrate 2820 and height Hl of gap-defining element 2830.

[0135] The design of the reagent dispensing element 2810 can provide a variety of advantageous functions. For example, the reagent dispensing element 2810 can include geometric features that can control fluid flow or movement as the substrates 2800 (and thus 2820) and 2835 are brought into or maintained in contact with each other within the sample handling apparatus described herein. Advantageously, a structure or finish of a surface of the reagent dispensing element 2810 can provide hydrophobicity. The surface can be formed to include a micro structure in the surface that can hold small volumes of air and thereby create a hydrophobic surface. In some embodiments, the hydrophobic surface of the reagent dispensing element 2810 can include one or more structures shaped like a lotus-leaf. A hydrophobic surface, pattern, finish, or coating on the reagent dispensing element 2810 can provide the benefit of confining the reagent medium 2815 within a desired location on the reagent dispensing element 2810 and/or providing a desired contact angle to reduce entrapment of bubbles between the reagent medium 2815 and the substrate 2835.

[0136] The reagent dispensing element 2810 can suppress fluid flow or movement as the substrates 2800 (and thus 2820) and 2835 are brought into or maintained in contact with each other within the sample handling apparatus described herein. The reagent dispensing element 2810 can trap or reduce bubbles from entering the array 2825 as the substrates 2800 (and thus 2820) and 2835 are brought into or maintained in contact with each other within the sample handling apparatus described herein. The reagent dispensing element 2810 can indicate to a user a suitable location for providing a reagent, such as a permeabilization reagent.

[0137] As shown in FIG. 9C, a third substrate 2835 can include a sample 2840 thereon. The sample handling apparatus described herein can be configured to bring the substrate 2835 and thus the sample 2840 into proximity with the array 2825 in a sandwich configuration as described herein. The reagent medium 2815 can be provided via the reagent dispensing element 2810 and can migrate toward the second substrate 2820 to permeabilize the sample 2840 and cause analytes of the sample 2840 to migrate to the array 2825 for spatial analysis.

[0138] FIGS. 10A-10C depict a sandwiching process using the substrates described in relation to FIGS. 9A-9C. In FIGS. 10A-10C, the sample 2840 has been excluded from substrate 2835 for clarity but would normally be included during the sandwiching process as described herein. As shown in FIG. 10A, a reagent 2815 has been dispensed atop the reagent dispensing element 2810 and the substrate 2835 is brought into initial sandwich configuration with the substrate 2800 (and 2820). In the embodiment shown in FIGS. 10A-10C, the reagent dispensing element 2810 can include a plurality of material layers 2905, e.g., two or more layers such as layers 2905A and 2905B. For example, a first layer 2905B can include a rigid material and a second layer 2905B can include a compressible material. In some embodiments, each layer 2905 can include a compressible material. In some embodiments, the layers 2905 can include materials that have a varying elasticity or compressibility. In some embodiments, the reagent dispensing element 2810 can include hydrophobic materials to aid dispersion of the reagent 2815 into a sealed chamber 2910 that can be formed by sandwiching the third substrate 2835 with respect to the first substrate 2800 (and the second substrate 2820). In some embodiments, the reagent dispensing clement 2810 can include a hydrophobic coating. For example, layer 2905A can be a hydrophobic coating and layer 2905B can be a compressible material. In some embodiments, a hydrophobic coating can be applied a top a first layer (e.g., layer 2905 A) of a reagent dispensing element 2810 including a plurality of material layers 2905. In some embodiments, the hydrophobic material can include a polymer, silicone, glass, or polydimethylsiloxane. In some embodiments, the first layer 2905A can include an adhesive, such as PET. Using an adhesive layer 2905A can provide a desired surface hydrophobicity, contact angle, and surface properties to reduce friction between the substrate 2835 and the reagent dispensing element 2810.

[0139] In some embodiments, the reagent dispensing element 2810 can be applied to the substrate 2800 using an adhesive. In some embodiments, the reagent dispensing element 2810 can be molded on to the substrate 2800.

[0140] As shown in FIG. 10B, a fluidically sealed chamber 2910 can be formed as the substrate 2835 is brought into contact with the reagent dispensing element 2810 during the sandwiching process. In some embodiments, the chamber 2910 can be fully sealed or partially sealed. The reagent 2815 can flow from the reagent dispensing element 2810 and into a cavity to surround the substrate 2820 and flow onto the array 2825. As shown in FIG. 10C, the fluidically sealed chamber 2910 can be maintained as the substrate 2835 is brought into further contact with the reagent dispensing element 2810 during the sandwiching process. For example, the reagent dispensing element 2810 is compressed after initial contact with the substrate 2835. As the reagent dispensing element 2810 is compressed downward (as shown by the arrows), the gap-defining elements 2830 can limit the vertical travel of the substrate 2835 so as to maintain a suitable gap height between the substrate 2820 (and the array 2825) and a sample present on the substrate 2835. In this way, a pre-configured gap height can be provided or maintained to enable permeabilization of the sample present on substrate 2835 and migration of analytes therein onto the array 2825.

[0141] As shown in FIGS. 11A and 1 IB, the gap-defining elements 2810 can be provided on the substrate 2820 in a variety of configurations. In the embodiment shown in FIG. 11 A, a plurality of gap-defining elements 2830 can be arranged at respective comers of the substrate 2820. In some embodiments, the gap-defining elements 2830 can be provided around the periphery of the substrate 2820. Although the gap-defining element 2830 shown in FIG. 11 A have a circular cross- sectional shape, a variety of non-limiting cross-sectional shapes can be envisioned, such as square shapes, elliptical shapes, rectangular shapes or the like. As shown in FIG. 11B, the gap-defining element 2830 can include a unibody structure formed at the periphery of the substrate 2820. In some embodiments, the gap-defining elements 2830 can be formed on the substrate 2800 alternatively to or in addition to forming the gap-defining elements 2830 on the substrate 2820.

[0142] A variety of non-limiting designs can be envisioned for the reagent dispensing element 2810 to aid reagent flow suppression and minimize bubble formation or air being trapped as the reagent 2815 is dispensed. Air can become trapped in gaps between the reagent dispensing element 2810 and the substrate 2820 during reagent dispensing, which can cause the reagent to flow, drift, contract, or expand undesirably. To mitigate this, the gap volume can be reduced, the gap volume can be increased, or portions of the gap can be pre wetted or prefilled, either partially or completely, before the sandwich configuration is finalized and the chamber 2910 is fluidically sealed. As shown in FIG. 12A, a plurality of gaps 3105 and 3110 can be formed between the reagent dispensing element 2810 and the second substrate 2820. As shown in FIG. 12A, the gap 3110 can be larger than the gaps 3105 such that the reagent 2815 fills gap 3110 first before gaps 3105. In some embodiments, one or more gaps 3105 can be larger (e.g., such as gap 3110) and the larger gap can be located at one or more locations or sides with respect to the substrate 2820.

[0143] As shown in FIG. 12B, the reagent dispensing element 2810 can include a channel 3115 that can be fluidically coupled to the gaps 3105. The reagent 2815 can fill the channel 3115 before filling the gaps 3105. As shown in FIG. 12C, the reagent dispensing element 2810 can include a cavity 3120. The cavity 3120 can be configured for a dual-dispensing operation in which both the gaps 3105 and the cavity 3120 are filled with reagent 2815 in parallel.

Methods of Forming Gap Defining Elements, Array Substrates, and Arrays

[0144] Also provided herein are methods of preparing a substrate comprising a gap-defining element. The methods may comprise forming a gap-defining element on the substrate from a rigid material. The gap-defining element can have a height configured to maintain a separation distance between a first substrate and a second substrate. Exemplary gap-defining element heights are described herein.

[0145] The gap-defining element 2830 can be formed by plating, coating, or deposition of a rigid material onto a substrate, such as substrates 2820 or 2800. In some embodiments, the gap-defining element 2830 can be printed onto a substrate, such as substrates 2820 or 2800. In some embodiments, a gap-defining element 2830 can be formed by molding a rigid material using a mold and/or a mold mask. In some embodiments, the gap-defining clement 2830 can be formed by curing a material using a photostimulus, such as ultraviolet light. The wavelength of light used to cure or develop the gap-defining element 2830 can vary depending on the type of material of the gap-defining element 2830. In some embodiments, a gap-defining element 2830 can be formed using lithography. Lithography can be advantageous for forming gap-defining elements 2830 with high precision geometries and/or tolerances compared to other methods. As such, lithography can be advantageous for forming gap-defining elements 2830 with heights less than 50 microns (e.g., 5 microns).

[0146] In some embodiments, the gap-defining element material may be or may comprise a photoresist material. A photoresist is a light-sensitive material used in processes (such as photolithography and photoengraving) to form a pattern on a surface. In some embodiments, the light-sensitive material can be used on photolithography and photoengraving processes to form a gap-defining element on a substrate. A photoresist may comprise a polymer, a sensitizer, and/or a solvent. The photoresist composition used herein is not limited to any specific proportions of the various components.

[0147] Photoresists can be classified as positive or negative. In positive photoresists, the photochemical reaction that occurs during light exposure weakens the polymer, making it more soluble to developer, so a positive pattern is achieved. In the case of negative photoresists, exposure to light causes polymerization of the photoresist, and therefore the negative photoresist remains on the surface of the substrate where it is exposed, and the developer solution removes only the unexposed areas. In some embodiments, the photoresist used herein is a positive photoresist. In some embodiments, the photoresist is degraded or removable with UV light.

[0148] In some embodiments, a photoresist composition can form a gap-defining element as described herein using a lithography process, such as a photolithography process. In some embodiments, a photoresist composition can form a micro or nanopattern on the gap-defining element via a lithography process, such as a photolithography process. In some embodiments, the lithography process uses a substrate material and a thin photoresist layer is formed on a substrate. The substrate is optionally baked to fix the photoresist layer on the substrate. In some embodiments, the photoresist layer on the substrate is exposed to radiation. The exposed photoresist layer can be treated with a developing solution, and by dissolving and removing the exposed area of the photoresist layer, a micro or nanopattern is formed. In some embodiments, a photolithography process disclosed herein may comprise forming a photoresist layer on a substrate using a photoresist composition; selectively exposing the photoresist layer; and developing the exposed photoresist layer. In some embodiments, a photolithography process disclosed herein comprises coating a photoresist composition on a substrate and drying (soft baking) the coated substrate. In some embodiments, a photolithography process disclosed herein comprises coating with a spin coater, a bar coater, a blade coater, a curtain coater, a screen printer or the like, and/or a spray coater or the like, and any method capable of coating a photoresist composition may be used. Drying (soft baking) of the substrate may be pre-formed under a suitable condition and may comprise, for example, an oven, a hot plate, vacuum drying and the like, but is not limited thereto. When going through the drying, a solvent is removed from the photoresist composition, increasing adhesive strength between the wafer and the photosensitive resin layer, and the photoresist layer may be secured on the substrate.

[0149] In some embodiments, the selectively exposing of the photoresist layer is performed by aligning a mask on the photoresist and exposing an area of the photoresist layer not covered by the mask to ultraviolet rays. The mask may be in contact with the photoresist layer or may also be aligned at a certain distance from the photoresist layer. In some embodiments, a light source irradiated as a light irradiation means may comprise electromagnetic waves, extreme ultraviolet rays (EUV), from ultraviolet rays to visible rays, an electron beam, X-rays, laser rays and the like. Known means such as a high-pressure mercury lamp, a xenon lamp, a carbon arc lamp, a halogen lamp, a cold cathode tube for a copier, an LED and a semiconductor laser may be used. In some embodiments, the selectively exposing of the photoresist layer may further comprise heating (postexposure baking) the exposed photoresist layer after the exposure. In some embodiments, developing of the exposed photoresist layer comprises removing the exposed portion in the photoresist layer by immersing in a developing solution. Any photoresist developing methods known in the art may be used and are not limited to a rotary spray method, a paddle method, or an immersion method accompanying ultrasonic treatment. Examples of the developing solution may comprise alkali metal or alkaline earth metal hydroxides, carbonates, hydrogen carbonates, an aqueous basic solution such as an ammonia water quaternary ammonium salt may be used. For instance, an aqueous ammonia quaternary ammonium solution such as an aqueous tetramethyl ammonium solution may be used. [0150] In some embodiments, the photoresist further comprises an acid scavenger. In some embodiments, the photoresist in the first and the second region comprises the same acid scavenger. In some embodiments, the photoresist in the first and the second region comprises different acid scavengers. In some embodiments, an acid scavenger acts to neutralize, adsorb and/or buffer acids, and may comprise a base or alkaline compound. In some embodiments, acid scavengers act to reduce the amount or concentration of protons or protonated water. In some embodiments, an acid scavenger acts to neutralize, diminish, or buffer acid produced by a photoacid generator. In some embodiments, an acid scavenger exhibits little or no stratification over time or following exposure to heat. In some embodiments, acid scavengers may be further subdivided into “organic bases” and “polymeric bases.” A polymeric base is an acid scavenger (e.g., basic unit) attached to a longer polymeric unit. A polymer is typically composed of a number of coupled or linked monomers. The monomers can be the same (to form a homopolymer) or different (to form a copolymer). In a polymeric base, at least some of the monomers act as acid scavengers. An organic base is a base which is joined to or part of a non-polymeric unit. Non-limiting examples of organic bases include, without limitation, amine compounds (e.g., primary, secondary and tertiary amines). Generally, any type of acid scavenger, defined here as a traditional Lewis Base, an electron pair donor, can be used in accordance with the present disclosure. The acid scavenger may be a tertiary aliphatic amine or a hindered amine.

[0151] In some embodiments, the photoresist comprises a quencher, such as a base quencher. The quencher that may be used in the photoresist composition may comprise a weak base that scavenges trace acids, while not having an excessive impact on the performance of the positive photoresist. Illustrative examples of quenchers that can be employed include, but are not limited to: aliphatic amines, aromatic amines, carboxylates, hydroxides, or combinations thereof and the like. Base quenchers may be used in photoresist formulations to improve performance by quenching reactions of photoacids that diffuse into unexposed regions. Base quenchers may comprise aliphatic amines, aromatic amines, carboxylates, hydroxides, or combinations thereof. Examples of base quenchers include but are not limited to, trioctylamine, 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU), 1 -piperidineethanol (1PE), tetrabutylammonium hydroxide (TBAH), dimethylamino pyridine, 7-diethylamino-4-methyl coumarin (Coumarin 1), tertiary amines, sterically hindered diamine and guanidine bases such as 1,8- bis(dimethylamino)naphthalene (PROTON SPONGE), berberine, or polymeric amines such as in the PLURONIC or TETRONIC series commercially available from BASF. In some embodiments, the photoresist in the first and the second region comprises the same base quencher. In some embodiments, the photoresist in the first and the second region comprises different base quenchers. [0152] In some embodiments, the photoresist further comprises a photosensitizer. A photosensitizer is a molecule that produces a chemical change in another molecule in a photochemical process. Photosensitizers are commonly used in polymer chemistry in reactions such as photopolymerization, photocrosslinking, and photodegradation. Photosensitizers generally act by absorbing ultraviolet or visible region of electromagnetic radiation and transferring it to adjacent molecules. In some embodiments, photosensitizer shifts the photo sensitivity to a longer wavelength of electromagnetic radiation. The sensitizer, also called a photosensitizer, is capable of activating the photoacid generator (PAG) at, for example, a longer wavelength of light in accordance with an aspect of the present disclosure. In some embodiments, the concentration of the sensitizer is greater than that of the PAG, such as 1.1 times to 5 times greater, for example, 1.1 times to 3 times greater the concentration of PAG. Examples of photosensitizer may include anthracene, N-alkyl carbazole, benzo [a]phenoxazine, and thioxanthone compounds. Exemplary sensitizers suitable for use in the methods disclosed herein include but are not limited to, isopropylthioxanthone (ITX), and lOH-phenoxazine (PhX). In some embodiments, the photoresist in the first and the second region comprises the same photosensitizer. In some embodiments, the photoresist in the first and the second region comprises different photosensitizers.

[0153] In some embodiments, the photoresist further comprises a matrix. The matrix generally refers to polymeric materials that may provide sufficient adhesion to the substrate when the photoresist formulation is applied to the top surface of the substrate and may form a substantially uniform film when dissolved in a solvent and deposited on top of a substrate. Examples of a matrix may include, but are not limited to, polyester, polyimide, polyethylene naphthalate (PEN), polyvinyl chloride (PVC), polymethylmethacrylate (PMMA) and polycarbonate, or a combination thereof. The matrix may be chosen based on the wavelength of the radiation used for the generation of acid when using the photoresist formulation, the adhesion properties of the matrix to the top surface of the substrate, the compatibility of the matrix to other components of the formulation, and the ease of removable or degradation after use. In some embodiments, the photoresist in the first and the second region comprises the same matrix. In some embodiments, the photoresist in the first and the second region comprises different matrices. [0154] In some embodiments, the photoresist further comprises a surfactant. Surfactants may be used to improve coating uniformity, and may include ionic, non-ionic, monomeric, oligomeric, and polymeric species, or combinations thereof. Examples of possible surfactants include fluorine- containing surfactants such as the FLUORAD series available from 3M Company in St. Paul, Minn., and siloxane-containing surfactants such as the SILWET series available from Union Carbide Corporation in Danbury, Conn. In some embodiments, the photoresist in the first and the second region comprises the same surfactant. In some embodiments, the photoresist in the first and the second region comprises different surfactants.

[0155] In some embodiments, the photoresist further comprises a casting solvent. A casting solvent may be used so that the photoresist may be applied evenly on the substrate surface to provide a defect-free coating. Examples of suitable casting solvents may include ethers, glycol ethers, aromatic hydrocarbons, ketones (e.g., methyl ethyl ketone), esters, ethyl lactate, y- butyrolactone, cyclohexanone, ethoxyethylpropionate (EEP), a combination of EEP and gammabutyrolactone (GBL), propylene glycol ethyl ether acetate, amyl acetate, propylene glycol methyl ether acetate (PGMEA), and combinations thereof. In some embodiments, the photoresist in the first and the second region comprises the same casting solvent. In some embodiments, the photoresist in the first and the second region comprises different casting solvents.

[0156] Methods of applying photoresist to the substrate include, but are not limited to, dipping, spreading, spraying, or any combination thereof. In some embodiments, the photoresist is applied via spin coating, thereby forming a photoresist layer on the substrate.

[0157] In some embodiments, the photoresist may be removed and re-applied. For example, the photoresist may be stripped from the substrate and/or the oligonucleotides ligated to the substrate. Removal of photoresist can be accomplished with various degrees of effectiveness. In some embodiments, the photoresist is completely removed from the substrate and/or the oligonucleotides ligated to the substrate before re-application. Methods of removing photoresist may include, but are not limited to, using organic solvent mixtures, using liquid chemicals, exposure to a plasma environment, or other dry techniques such as UV/03 exposure. In some embodiments, the photoresist is stripped using organic solvent. In some embodiments, the photoresist may be removed after each cycle of in situ array generation and re-applied prior to the next cycle of in situ array generation. In some embodiments, the photoresist removed, and the photoresist re-applied prior to the next cycle is the same photoresist. In some embodiments, the photoresist removed, and the photoresist re-applied prior to the next cycle are different photoresists.

[0158] In these processes, a photoresist is a light-sensitive material used to form a pattern on a surface. A photoresist may comprise a polymer, a sensitizer, and/or a solvent. The photoresist composition used herein is not limited to any specific proportions of the various components.

[0159] In some embodiments, one or more photomasks may be used to selectively remove photoresist on the substrate. The mask is designed in such a way that the exposure sites can be selected, and thus specify the coordinates on the array where each nucleotide can be attached. The process can be repeated, a new mask is applied activating different sets of sites and coupling different bases, allowing arbitrary oligonucleotides to be constructed at each site. This process can be used to synthesize hundreds of thousands or millions of different oligonucleotides. In some embodiments, the substrate is irradiated through a patterned mask. The mask may be an opaque plate or film with transparent areas that allow light to shine through in a pre-defined pattern. After the irradiation step, the mask may be removed, translated to a different region on the substrate, or rotated. In some embodiments, a different photomasking pattern may be used in each barcoding round. In some embodiments, the same photomasking pattern may be used in each barcoding round. Using a series of photomasks, photoresist in desired regions of the substrate may be iteratively irradiated and subsequently removed.

[0160] The material of the photomask used herein may comprise silica with chrome in the opaque part. For example, the photomask may be transparent fused silica blanks covered with a pattern defined with a chrome metal absorbing film. The photomask may be used at various irradiation wavelengths, which include but are not limited to, 365 nm, 248 nm, and 193 nm.

[0161] In preferred embodiments, the gap-defining element 2830 can be formed via photopatterning or photolithography methods. The photo-patterning or photo-lithography methods can utilize a photoresist material, such as a positive photoresist material or a negative photoresist material to form the gap-defining element 2830 described herein. FIG. 13 is a diagram illustrating a method of forming a gap-defining element 2830 configured for use with the sample handling apparatus described herein using a positive photoresist material in accordance with some example implementations. As shown in step A, a substrate can be provided, such as substrate 2820. In step B, a positive photoresist material 3200 can be coated onto the substrate 2820. In some embodiments, the photoresist material can be spin coated onto the substrate 2820. At step C, an photo-stimulus or light source 3205, such as an ultraviolet light source can be provided to illuminate the positive photoresist material 3200. Exposing the array area 3210 with the light source 3205 can solubilize the positive photoresist material 3200 and leaving the gap-defining element 2830 formed from the unexposed positive photoresist material 3200 as shown in step D. [0162] FIG. 14 is a diagram illustrating a method of forming a gap-defining element 2830 configured for use with the sample handling apparatus described herein using a negative photoresist material in accordance with some example implementations. In some embodiments, the gap-defining element 2830 can be formed using a negative photoresist material instead of a positive photoresist material as described in relation to FIG. 13. In FIG. 14, as shown in step A, a substrate can be provided, such as substrate 2820. In step B, a negative photoresist material 3300 can be coated onto the substrate 2820. In some embodiments, the photoresist material can be spin coated onto the substrate 2820. At step C, a photo-stimulus or light source 3305, such as an ultraviolet light source can be provided to illuminate the negative photoresist material 3300. Exposing a location of the gap-defining element 2830 with the light source 3305 can cross-link the negative photoresist material 3300 in that location and can develop the non-crosslinked area of the array 2825. The gap-defining element 2830 is formed from the exposed negative photoresist material 3300 as shown in step D.

[0163] FIG. 15 is a process flow diagram illustrating an example process 3400 for forming a plurality of second substrates as described herein, each second substrate including a plurality of array elements and at least one gap-defining element as described herein. In some embodiments, the second substrate can also include a spacer, a spacer boundary, a handling area, and/or at least one array fiducial in accordance with example implementations described herein. The second substrates formed by process 3400 can include a gap-defining element formed thereon according to the methods described herein.

[0164] In operation 3410, an array base substrate including a first surface can be provided. In some embodiments, the array base substrate can be a glass slide or glass wafer suitable for configuring a plurality of arrays thereon and capable of being diced or cut so that multiple individual second substrates can be formed from the array base substrate. In some embodiments, the array base substrate can be -0.5 mm thick. The array base substrate can be 6 or 8 inches in diameter. Hundreds of arrays can be formed on the first surface of array base substrate right next to each other.

[0165] In operation 3420, a plurality of arrays 2825 can be formed on the first surface of the array base substrate. Each array 2825 can include a plurality of array elements and at least one gap-defining element 2830. The plurality of arrays can be formed on the array base substrate as described in relation to operation 3530 of FIG. 16.

[0166] In operation 3430, the array base substrate including the plurality of arrays formed thereon can be diced or cut to form one or more second substrates 2820. Each of the second substrates 2820 can include at least one array 2825. The at least one array 2825 on the individually formed second substrates 2820 can include the plurality of array elements and at least one gapdefining element 2825.

[0167] FIG. 16 is a process flow diagram illustrating an example process 3500 for forming an array substrate according to some implementations of the current subject matter. The process 3500 can be described in relation to the array substrate 2820 shown and described in relation to FIGS. 9A-9C. In operation 3510, a first substrate including a first surface can be provided. The first substrate can correspond to substrate 2800. In operation 3520, a second substrate including a first surface can be provided. The second substrate can correspond to substrate 2820. The first surface of the second substrate can include an array 2825. The first surface of the second substrate can be dimensioned to receive one or more gap-defining elements, such as gap-defining element 2830, to surround the array 2825. The gap-defining element 2830 can have a height configured to provide or maintain a gap between the second substrate 2820 and the third substrate, e.g., the tissue substrate, 2835.

[0168] In operation 3530, an array 2825 can be formed on the first surface of the second substrate 2820. In some embodiments, the array can be formed on the first surface of the second substrate using photolithography or photoengraving processes as described above in regard to forming gapdefining elements 2830 on the array substrate 2820.

[0169] In some aspects, the method provided herein comprises attaching oligonucleotides (e.g. a barcode) to a substrate. Oligonucleotides may be attached to the substrate according to the methods set forth in U.S. Patent Nos. 6,737,236, 7,259,258, 7,375,234, 7,427,678, 5,610,287, 5,807,522, 5,837,860, and 5,472,881; U.S. Patent Application Publication Nos. 2008/0280773 and 2011/0059865; Shalon et al. (1996) Genome Research, 639-645; Rogers et al. (1999) Analytical Biochemistry 266, 23-30; Stimpson et al. (1995) Proc. Natl. Acad. Sci. USA 92, 6379-6383; Beattie et al. (1995) Clin. Chem. 45, 700-706; Lamturc et al. (1994) Nucleic Acids Research 22, 2121-2125; Beier et al. (1999) Nucleic Acids Research 27, 1970-1977; Joos et al. (1997) Analytical Biochemistry 247, 96-101; Nikiforov et al. (1995) Analytical Biochemistry 227, 201- 209; Timofeev et al. (1996) Nucleic Acids Research 24, 3142-3148; Chrisey et al. (1996) Nucleic Acids Research 24, 3031-3039; Guo et al. (1994) Nucleic Acids Research 22, 5456-5465; Running and Urdea (1990) BioTechniques 8, 276-279; Fahy et al. (1993) Nucleic Acids Research 21, 1819-1826; Zhang et al. (1991) 19, 3929-3933; and Rogers et al. (1997) Gene Therapy 4, 1387-1392. The entire contents of each of the foregoing documents are incorporated herein by reference.

[0170] Arrays, such as array 2825, can be prepared by a variety of methods. In some embodiments, arrays are prepared through the synthesis (e.g., in situ synthesis) of oligonucleotides on the array, or by jet printing or lithography. For example, light-directed synthesis of high-density DNA oligonucleotides can be achieved by photolithography or solid-phase DNA synthesis. To implement photolithographic synthesis, synthetic linkers modified with photochemical protecting groups can be attached to a substrate and the photochemical protecting groups can be modified using a photolithographic mask (applied to specific areas of the substrate) and light, thereby producing an array having localized photo-deprotection. Many of these methods are known in the art, and are described e.g., in Miller et al., “Basic concepts of microarrays and potential applications in clinical microbiology.” Clinical microbiology reviews 22.4 (2009): 611-633; US201314111482A; US9593365B2; US2019203275; and WO2018091676, which are incorporated herein by reference in their entirety.

[0171] Returning to FIG. 16, in operation 3540, the gap-defining element 2830 can be formed on the first surface of the second substrate. In some embodiments, the gap-defining element 2830 can surround the array 2825 (as shown in FIG. 1 IB). Applying the gap-defining element 2830 to the first surface of the second substrate 2820 can include forming the gap-defining element 2830 on the first surface of the second substrate 2820 as described in relation to FIGS. 13 and 14.

[0172] In operation 3550, the second substrate 2820 can be applied to the first surface of the first substrate 2800 to form the array substrate 2845 including both the first substrate 2800 and the second substrate 2820.

Experimental Data for Forming Gap Defining Elements, Array Substrates, and Arrays [0173] As shown in the top-down view of FIG. 17A, a workflow 3600 is shown depicting steps A-C of a method of forming a substrate including gap defining elements and a reagent dispensing area as described herein. FIG. 17B is a side-view of the same workflow 3600. A substrate 2820 can be formed to include a gap-defining element 2830 positioned outside an array 2825. The gapdefining element 2830 can have a thickness of 5 microns and a width of 250 microns. The gapdefining element 2830 can be configured around the edge of the substrate 2820 using a photoresist material as shown in step A.

[0174] The substrate 2820 can be about 7.75 mm x 7.75 mm and can be diced from a wafer having a thickness of about 0.75 mm. The diced substrate 2820 can be placed atop a substrate 2800, such as a glass slide as shown in step A.

[0175] In step B, a compressible reagent dispensing area 2810 can be formed onto the substrate 2800. The reagent dispensing area 2810 can be formed by adhering a first material 2905 A in a first layer onto the substrate 2800. The first material 2905A can include a compressible material made about 0.8 mm thick that was laser cut to the dimensions of 24 mm long x 15 mm wide and includes a 9 mm x 9 mm window cut out therefrom. The window can be sized to correspond to the area of the substrate 2820. As shown in step C, the first material 2905A can be applied to the substrate 2800 and a second material 2905B can be overlaid atop the first material 2905A to form the reagent dispensing area 2810. The second material 2905B can be 5 microns thick and can be a soft polyethylene terephthalate.

[0176] As shown in FIGS. 18A-18C, a sandwiching workflow 3700 can be performed to assess the fluidics of the system. In this experiment, three sample substrates (e.g., substrate 2800 configured with substrate 2820 and reagent dispensing area 2810 thereon) were tested in three different sample handling apparatuses as described herein. In the pre- sandwiching configuration shown in FIG. 18A, a 25 microliter volume of reagent medium 2815 was provided onto the reagent dispensing area 2810 and a dye spot 3705 is added to the surface of the substrate 2820 to evaluate fluid flow and diffusion during sandwiching.

[0177] As shown in FIG. 18B, the substrate 2835 can be brought into initial contact with the reagent dispensing area 2810 to form the fluidically sealed chamber 2910. As shown in FIG. 18C, the sandwiching operation can be completed so that the reagent dispensing area 2810 is compressed and the gap-defining elements 2830 come into contact with the substrate 2835 thereby maintaining a predetermined height of the fluidically sealed chamber 2910 and a predetermined gap distance between the substrates 2820 and 2835.

[0178] Flow speed was measured for each sample substrate by tracking the center of the dye spot 3705 as it diffuses during the sandwiching configuration shown in FIG. 18C. When the center of the dye spot 3705 is stationary it can be understood that no or low flow is present, and thus, fluid flow is being suppressed. For example, as shown in FIG. 19A, the peak of the Gaussian fit can indicate the extent of diffusion of the dye spot 3705 imaged in FIG. 19B. As shown in FIG. 19C, flow speed can be determined over a 30 minute period. Initially, the dye spot 3705 can diffuse, until about 5 minutes, after which, the sample substrate reduced and nearly eliminated further diffusion, thereby enhancing flow suppression. FIGS. 20A-20F depict image data of diffusion of the dye spot 3705 taken at 5 minute intervals over the 30 minute period for one of the sample substrates formed by the workflow of FIGS 17A-17C and sandwiched using the workflow 3700 of FIGS. 18A-18C. As shown in FIGS. 20A-20F, the dye spots show little to no diffusion over the experimental period as flow is suppressed.

[0179] As shown in FIGS. 21 A-21C, flow rates of the three sample substrates were measured in two passes (e.g., A and B) on each of the three sample handling apparatuses. FIG. 21 A corresponds to evaluation of the three sample substrates on the first sample handling apparatus. FIG. 21B corresponds to evaluation of the three sample substrates on the second sample handling apparatus. FIG. 21C corresponds to evaluation of the three sample substrates on the third sample handling apparatus. Plots of the flow rate of the dye spot 3705 was measured in micrometers/second over the 30 minute experimental period were generated as shown in FIGS 21A-21C.

[0180] The plots illustrate limited flow during sandwich holding (e.g., the configuration corresponding to FIG. 18C) and evidence that the reagent dispensing area 2810 successfully compressed down to the thickness of the substrate 2820 and the gap-defining element 2830. Additionally, the plots illustrate that flow is suppressed over time by the gap-defining element 2830 providing a hard stop for the substrate 2835 during sandwiching with substrates 2800 and 2820. Passing test inns were considered to have a flow speed of less than 0.5 micrometers/second across the 30 minute sandwiching period and excluded the first two minutes of sandwiching. The initially higher flow rates shown in the plots (e.g., about 1.5 micrometers/second during the first minutes of sandwiching) were caused by inertia created and imparted onto the flow during closing of the sample handling apparatus in the initial sandwiching configuration corresponding to FIG. 18B.

[0181] One or more aspects or features of the subject matter described herein may be realized in digital electronic circuitry, integrated circuitry, specially designed ASICs, field programmable gate arrays (FPGAs) computer hardware, firmware, software, and/or combinations thereof. These various aspects or features may include implementation in one or more computer programs that are executable and/or interpretable on a programmable system including at least one programmable processor, which may be special or general purpose, coupled to receive data and instructions from, and to transmit data and instructions to, a storage system, at least one input device, and at least one output device. The programmable system or computing system may include clients and servers. A client and server are generally remote from each other and typically interact through a communication network. The relationship of client and server arises by virtue of computer programs running on the respective computers and having a client-server relationship to each other.

[0182] These computer programs, which may also be referred to as programs, software, software applications, applications, components, or code, include machine instructions for a programmable processor, and may be implemented in a high-level procedural and/or object-oriented programming language, and/or in assembly /machine language. As used herein, the term “machine-readable medium” refers to any computer program product, apparatus and/or device, such as for example magnetic discs, optical disks, memory, and Programmable Logic Devices (PLDs), used to provide machine instructions and/or data to a programmable processor, including a machine-readable medium that receives machine instructions as a machine-readable signal. The term “machine-readable signal” refers to any signal used to provide machine instructions and/or data to a programmable processor. The machine-readable medium may store such machine instructions non-transitorily, such as for example as would a non-transient solid-state memory or a magnetic hard drive or any equivalent storage medium. The machine-readable medium may alternatively or additionally store such machine instructions in a transient manner, such as for example, as would a processor cache or other random access memory associated with one or more physical processor cores.

[0183] To provide for interaction with a user, one or more aspects or features of the subject matter described herein may be implemented on a computer having a display device, such as for example a cathode ray tube (CRT) or a liquid crystal display (LCD) or a light emitting diode (LED) monitor for displaying information to the user and a keyboard and a pointing device, such as for example a mouse or a trackball, by which the user may provide input to the computer. Other kinds of devices may be used to provide for interaction with a user as well. For example, feedback provided to the user may be any form of sensory feedback, such as for example visual feedback, auditory feedback, or tactile feedback; and input from the user may be received in any form, including acoustic, speech, or tactile input. Other possible input devices include touch screens or other touch-sensitive devices such as single or multi-point resistive or capacitive track pads, voice recognition hardware and software, optical scanners, optical pointers, digital image capture devices and associated interpretation software, and the like.

[0184] The subject matter described herein may be embodied in systems, apparatus, methods, and/or articles depending on the desired configuration. The implementations set forth in the foregoing description do not represent all implementations consistent with the subject matter described herein. Instead, they are merely some examples consistent with aspects related to the described subject matter. Although a few variations have been described in detail above, other modifications or additions are possible. In particular, further features and/or variations may be provided in addition to those set forth herein. For example, the implementations described above may be directed to various combinations and subcombinations of the disclosed features and/or combinations and subcombinations of several further features disclosed above. In addition, the logic flows depicted in the accompanying figures and/or described herein do not necessarily require the particular order shown, or sequential order, to achieve desirable results. Other implementations may be within the scope of the following claims.

Claims

1. A substrate for spatial analysis of a biological sample comprising: a first substrate comprising a first surface; a second substrate positioned atop the first surface of the first substrate, the second substrate comprising an array area on a first surface of the second substrate, the array area comprising an array of capture probes, at least one capture probe of the array of capture probes comprising a capture domain; at least one gap-defining element extending vertically from the first surface of the second substrate; and a reagent dispensing element on the first surface of the first substrate, the reagent dispensing element surrounding the array area of the second substrate and the at least one gapdefining element.

2. The substrate of claim 1, wherein the capture probe further comprises a spatial barcode, a unique molecular identifier (UMI), one or more functional domains comprising a primer binding site, or a combination thereof.

3. The substrate of claim 1, wherein the at least one gap-defining element surrounds the array area.

4. The substrate of any one of claims 1-3, wherein the at least one gap-defining element has a height of about 50 microns or less.

5. The substrate of any one of claims 1-4, wherein the at least one gap-defining element has a height of less than 2 microns, or has a height of 1 .5 microns, or has a height of about 1 micron.

6. The substrate of any one of claims 1-5, wherein a second gap-defining element extends vertically from the first surface of the first substrate.

7. The substrate of any one of claims 1 -6, further comprising a third substrate opposite the first surface of the first substrate, wherein the at least one gap-defining element is configured to maintain a gap height between the first substrate and the third substrate.

8. The substrate of claim 7, wherein the third substrate comprises a biological sample.

9. The substrate of claim 7 or 8, wherein the first substrate and the third substrate form a fluidically sealed chamber there between when the reagent dispensing element is compressed between the first substrate and the third substrate.

10. The substrate of claim 7, wherein the array area of the second substrate, the at least one gap-defining element, and the third substrate form a fluidically sealed chamber.

11. The substrate of any one of claims 1-10, wherein the at least one gap-defining element comprises a first material and the reagent dispensing area comprises at least one second material different than the first material.

12. The substrate of claim 11, wherein the at least one first material comprises a positive photoresist material.

13. The substrate of claim 11, wherein the at least one first material comprises a negative photoresist material.

14. The substrate of any one of claims 11-13, wherein the reagent dispensing element comprises a plurality of second materials arranged in a plurality of layers.

15. The substrate of claim 11 , wherein the at least one second material comprises a hydrophobic material and/or a material comprising reduced friction properties.

16. The substrate of claim 15, wherein the hydrophobic material comprises a polymer, silicone, glass, or polydimethylsiloxane.

17. The substrate of any one of claims 1 -16, wherein the reagent dispensing element is configured to control an amount of a fluid dispersed between the first substrate and the third substrate comprising a biological sample, suppress a flow of a fluid present between the first substrate and the third substrate comprising a biological sample as the first substrate and the third substrate are brought into contact with the reagent dispensing element, reduce bubble formation in a fluid present between the first substrate and the third substrate comprising a biological sample as the first substrate and the third substrate are brought into contact with the reagent dispending element, or identify a reagent dispensing area configured on the second substrate.

18. The substrate of any one of claims 1-17, further comprising a plurality of gap-defining elements, each gap-defining element positioned at a respective corner of the second substrate.

19. The substrate of any one of claims 1-18, wherein the first substrate is a glass slide.

20. A substrate for spatial analysis of a biological sample comprising: a first substrate having a first surface; and a second substrate coupled to the first surface of the first substrate, the second substrate comprising an array area on a first surface of the second substrate, wherein the array area comprises an array of features, wherein (i) a first feature of the array comprises one or more first capture probes, the one or more first capture probes comprising a first capture domain and a first spatial barcode that identifies a location of the first feature, and (ii) a second feature of the array comprises one or more second capture probes, the one or more second capture probes comprising a second capture domain and a second spatial barcode that identifies a location of the second feature, and (iii) the first feature or the second feature has a diameter, or a maximum distance between adjacent first features or second features, of 500 nm to 2 pm, 1 pm to 3 pm, 1 pm to 5 pm, or 1 pm to 10 pm; at least one gap-defining element extending vertically from the first surface of the second substrate; and a reagent dispensing element on the first surface of the first substrate, the reagent dispensing element surrounding the array area on the first surface of the second substrate and having a height of 750 microns or more.

21. The substrate of claim 20, wherein the at least one gap-defining element has a height of less than 50 microns, less than 40 microns, less than 30 microns, less than 20 microns, less than 10 microns, less than 5 microns, or less than 2 microns, or has a height of about 1.5 microns, or has a height of about 1 micron.

22. A method for spatial analysis of a biological sample, the method comprising: providing a sample handling apparatus holding a first substrate, a second substrate, and a reagent medium, wherein the first substrate comprises a biological sample mounted thereon, the biological sample comprising an analyte, the second substrate comprises a reagent dispensing element and a third substrate comprising an array comprising a plurality of capture probes and at least one gap-defining element, wherein at least one capture probe of the plurality of capture probes comprises a capture domain; aligning the first substrate with the second substrate, wherein the aligning assembles a chamber comprising the first substrate, the second substrate, the biological sample, and the reagent dispensing element, wherein the reagent dispensing element is positioned to at least partially surround or fully surround an area on the first substrate on which the biological sample is disposed and/or the array disposed on the third substrate, wherein the area of the first substrate, the reagent dispensing element, and the second substrate at least partially encloses a volume comprising the biological sample and the at least one gap-defining element maintains a gap height between the first substrate and the second substrate as the reagent dispensing element is compressed between the first substrate and the second substrate once the chamber is assembled; and releasing the analyte from the biological sample when the first substrate and second substrates are aligned, the releasing causing the analyte to migrate from the biological sample to the array.

23. The method of the claim 22, wherein the migrated analyte hybridizes to the at least one capture probe of the plurality of capture probes.

24. The method of any one of claims 22-23, wherein the at least one capture probe of the plurality of capture probes further comprises a spatial barcode, a unique molecular' identifier (UMI), one or more functional domains comprising a primer binding site, or a combination thereof.

25. The method of any one of claims 22-24, wherein the analyte migrates to the array via passive diffusion.

26. The method of any one of claims 22-25, wherein the analyte comprises a nucleic acid, optionally wherein the nucleic acid is DNA, RNA, or a ligated RTL probe.

27. The method of any one of claims 22-26, wherein the reagent medium comprises a permeabilization agent.

28. The method of claim 27, wherein the reagent medium comprises a protease, a detergent, or an RNAse.

29. The method of any one of claims 22-28, wherein the sample handling apparatus comprises a first member that receives the first substrate, a second member that receives the second substrate, and an adjustment mechanism configured to align the first substrate and the second substrate to assemble the chamber comprising the first substrate, the second substrate, the biological sample, and the reagent dispensing element.

30. The method of any one of claims 22-29, wherein the biological sample and the capture probe are contacted with the reagent medium when the first substrate is aligned with the second substrate.

31. The method of any one of claims 24-30, further comprising generating a barcoded polynucleotide comprising a sequence of the analyte or a sequence of a complement of the analyte, and the spatial barcode of the capture probe or a complement thereof, optionally wherein the generating the barcoded polynucleotide comprises extending the at least one capture probe using the analyte as a template, thereby generating an extended capture probe, and optionally amplifying the extended capture probe.

32. The method of claim 31, further comprising determining the sequence of the analyte or the complement thereof comprised in the barcoded polynucleotide.

33. The method of claim any one of claims 22-32, further comprising determining a location of the analyte within the biological sample based on the determined sequences of the analyte and spatial barcode comprised in the barcoded polynucleotide.

34. A method of preparing a substrate for use in spatial analysis of a biological sample, the method comprising: providing a first substrate comprising a first surface; providing a second substrate comprising a first surface, the second substrate comprising an array area comprising an array of capture probes, wherein at least one capture probe of the array of capture probes comprises a capture domain; applying the second substrate onto the first surface of the first substrate; and forming a reagent dispensing element on the first substrate surrounding the array area of the second substrate.

35. The method of claim 34, wherein providing the second substrate comprises: providing an array base substrate comprising a first surface; forming a plurality of arrays on the first surface of the array base substrate, each array of the plurality of arrays comprising a plurality of array elements; forming at least one gap-defining element on the first surface of the array base substrate for each array of the plurality of arrays, the at least one gap-defining element extending vertically from the first surface of the array base substrate; and dicing the array base substrate to form one or more second substrates, each second substrate of the one or more second substrates comprising at least one array comprising the plurality of array elements, and the at least one gap-defining element.

36. The method of claim 35, wherein the at least one gap-defining element surrounds the array area.

37. The method of claim 35 or 36, wherein the at least one gap-defining element extends vertically from the first surface of the first substrate.

38. The method of any one of claims 35-37, wherein the at least one gap-defining element is formed from a first material and the reagent dispensing element is formed from at least one second material.

39. The method of claim 38, wherein the first material is a positive photoresist material and forming the at least one gap-defining element on the first substrate or the second substrate comprises exposing the positive photoresist material to ultraviolet light at a location on the first substrate or the second substrate corresponding to the at least one gap-defining element, and applying a developer solution to remove the positive photoresist material from the location on the first substrate or the second substrate corresponding to the at least one gapdefining element.

40. The method of claim 38, wherein the first material is a negative photoresist material and forming the at least one gap-defining element on the first substrate or the second substrate comprises exposing the negative photoresist material to ultraviolet light at a location on the first substrate or the second substrate corresponding to a location of the at least one gap-defining element, and applying a developer solution to remove the negative photoresist material from the location on the first substrate or the second substrate corresponding to the at least one gapdefining element.

41. The method of any one claims 34-40, wherein forming the reagent dispensing element comprises forming a plurality of layers using a plurality of second materials.

42. The method of claim 38, wherein the at least one second material comprises a hydrophobic material.

43. The method of claim 42, wherein the hydrophobic material comprises a polymer, silicone, glass, or polydimethylsiloxane.

44. The method of any one of claims 34-43, further comprising providing a third substrate opposite the first surface of the first substrate, wherein the at least one gap-defining element is configured to maintain a gap height between the first substrate and the third substrate.

45. The method of claim 44, wherein the third substrate comprises a biological sample.

46. The method of claim 45, wherein the biological sample comprises a fresh and/or frozen tissue section or a fixed tissue section.

47. The method of claim 44, wherein the first substrate and the third substrate form a fluidically sealed chamber there between when the reagent dispensing element is compressed between the first substrate and the third substrate.

48. The method of any one of claims 35-47, wherein forming the at least one gap-defining element comprises forming the at least one gap defining element to have a height of about 50 microns or less.

49. The method of any one of claims 35-47, wherein the at least one gap-defining element has a height of less than 2 microns, or has a height of about 1.5 microns, or has a height of about 1 micron.

50. The method of any one of claims 34-49, wherein the reagent dispensing element is configured to control an amount of a fluid dispersed between the first substrate and the third substrate, suppress a flow of a fluid present between the first substrate and the third substrate as the first substrate and the third substrate are brought into contact with the reagent dispensing element, reduce bubble formation in a fluid present between the first substrate and the third substrate as the first substrate and the third second substrate are brought into contact with the reagent dispensing element, or identify a reagent dispensing area configured on the second substrate.

51. The method of claim any one of claims 34-50, further comprising forming a plurality of gap-defining elements, each gap-defining element formed at a respective comer of the second substrate.