WO2022098810A1

WO2022098810A1 - Assay support devices

Info

Publication number: WO2022098810A1
Application number: PCT/US2021/057972
Authority: WO
Inventors: David Maurice Cox
Original assignee: 10X Genomics, Inc.
Priority date: 2020-11-06
Filing date: 2021-11-04
Publication date: 2022-05-12
Also published as: CN217033310U

Abstract

A support device includes a substrate holder including a cover configured to receive a gasket, a base configured to receive a sample substrate, and a first locking tab and a second locking tab. The first and second locking tabs includes moveable tabs coupled to opposing first side walls of one of the cover and the base and non-moveable tabs coupled to opposing first side walls of the other of the cover and the base. The first locking tab has a length greater than a length of the second locking tab. The first locking tab extends along a length of the gasket. The moveable tabs are configured to engage with the non-moveable tabs to releasably secure the cover to the base.

Description

ASSAY SUPPORT DEVICES

BACKGROUND

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 crosstalk with other cells in the tissue.

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).

Furthermore, imaging systems used on spatial analyte data are inherently variable in their resolution and sensitivity. This is due in large part to the variability of manufacturers for imaging system components in addition to the arrangement of the imaging apparatus, differences between various types of imaging apparatuses, and image acquisition software. The image quality is further impacted by alterations in the image acquisition performed by the user. This problem becomes more apparent when trying to image samples of an unknow n fluorescent intensity or by having samples imaged by users of varying experience.

Moreover, in a laboratory environment, a variety of processing protocols are used to prepare a sample for analysis. These protocols can be performed in test tubes, on slides, or more generally, on a sample that is supported by a substrate. Certain protocols are performed at stable, controlled temperatures to ensure the fidelity of the sample and protocol reagents. Other protocols involve temperature cycling and other steps in which the temperature of the sample is adjusted in a controlled fashion. To heat the sample and its supporting substrate during a protocol, a thermocycler, heating plate, or other heating device may be used. As one example, thermocyclers can be as part of polymerase chain reaction protocols for nucleic acid amplification and in transcription and reverse transcription analytical sequences. Controlled heating of samples in thermocyclers and other heating devices also can occur to facilitate temperature-sensitive reactions for restriction enzyme digestion and rapid diagnostics, for example. In addition, a biological sample may be placed on a solid support to be analyzed for identification or characterization of an analyte, such as DNA, RNA, or other genetic material, within the sample. Printed guides may help improve placement of a sample on a solid support.

Current methods and devices to support biological samples (e.g., during spatial analysis and/or heating) often lack portability, do not provide access to specific sample regions or wells, and/or do not provide a vapor-tight seal for preventing cross-contamination between the sample regions or wells.

SUMMARY

Embodiments disclosed below include support devices for substrates including a sample region and methods of incubating a sample disposed on a sample region of a substrate. In some embodiments, a distinct advantage of the support devices and methods of the present disclosure is its construction as a one-piece device that provides ease of use to the user. For example, the one-piece design facilitates set-up and reduces time spent by the user in assembling the device and substrate. In some embodiments, the user can easily insert a substrate into the devices described without fastening multiple pieces. In some embodiments, the user does not use any tools to aid in the insertion of the substrate into the device or to aid in removing the substrate from the device. For example, the user may not need to use any type of fastener to assemble the support device or to secure a substrate to the support device, thereby providing a more efficient way of supporting a biological sample. In other examples, the devices disclosed herein may alternatively use one or more fasteners to assemble the support device and/or to secure a substrate to the support device.

In some embodiments, another distinct advantage of the support devices and methods of the present disclosure is its design that provides access to a specific sample region in a substrate via alignment of the plurality of apertures in the substrate holder and the plurality of openings in the gasket. In some embodiments, an additional advantage of the support devices and methods of the present disclosure is the uniform pressure applied to the substrate via the gasket and/or the rib or the plurality of ribs when the support device is in a closed position. Furthermore, a vapor-tight and/or air-tight seal is formed between the gasket and the substrate when the substrate holder is in the closed position. Such vapor-tight and/or air-tight seal can prevent transport of fluid between the plurality of openings of the gasket. The vapor-tight and/or air-tight seal can further prevent cross-contamination of biological samples via, e.g., the leakage of solution or fluid from a first sample region to a second sample region. Yet another advantage of the support devices and methods described in this disclosure, in some embodiments, is the minimization of shear forces on the substrate, for example, on the area of the substrate in proximity to the hinge or hinges. The minimization of shear forces can prevent damage to the substrate (e.g., breakage or cracking).

The devices provided herein can provide consistent and even heating to a substrate surface. Even heating can be critical to ensuring that preparative reactions performed on a sample supported by the substrate occur according to established protocols and achieve desired outcomes.

Further, heating a substrate (e.g., a glass slide) to temperatures above room temperature can cause condensation to form on an upper surface of an enclosed substrate well if the substrate is heated without an upper lid. Condensation can change the composition of reaction mixtures in the substrate wells, inhibiting preparative reactions, and/or producing unpredictable results. The devices described in this disclosure can be used to reduce or prevent condensation from forming in substrate wells via the vapor-tight and/or air-tight seal produced by the gasket.

Certain types of thermocyclers and heating devices are purpose-built for particular types of substrates such as multi-well substrates. Loading other types of substrates such as standard microscope slides into such devices can lead to uneven substrate heating. The devices described in this disclosure can be used to support substrates within heating devices that are not designed for such substrates, ensuring that adequate and even heat transfer occurs to the substrates. In particular, the devices can be used to adapt thermocyclers designed to accept multi-well substrates so that other types of substrates can be effectively heated within the thermocyclers as part of a sample preparation protocol.

In some embodiments, the devices of the disclosure allow a surface of the substrate to directly contact the surface of a heating device (e.g., a thermocycler), thereby permitting uniform heating throughout the substrate. That is, in some embodiments, no additional substrates or housing elements are required to be positioned in between the heat source and the substrates to be heated. In addition, because the devices of the disclosure allow the surface of the substrate to directly contact the surface of a heating device (e.g., a thermocycler), the temperature of the substrate can be more easily controlled by the user and can be heated to a desired temperature in less time than when using devices that do not allow surface contact between the substrate and the heating device (e.g., a thermocycler). Thus, samples (e.g., a biological samples) on the substrate can be heated uniformly and in a controlled manner. In one aspect, this disclosure is directed to a support device including: a substrate holder including: a cover including opposing first side walls, the cover configured to receive a gasket; a base including opposing first side walls, the base configured to receive a sample substrate; and a first locking tab and a second locking tab, the first locking tab and the second locking tab including moveable tabs coupled to the first side walls of one of the cover and the base and non-moveable tabs coupled to the first side walls of the other of the cover and the base, the first locking tab having a length greater than a length of the second locking tab, wherein the first locking tab extends along a length of the gasket, and wherein the moveable tabs are configured to engage with the non-moveable tabs to releasably secure the cover to the base.

In some embodiments, a height of the first locking tab is about equal to a height of the gasket. In some embodiments, the length of the first locking tab is about equal to the length of the gasket. In some embodiments, the device further includes at least one hinge and the at least one hinge extends from one of the second side walls of the cover to one of the second side walls of the base. In some embodiments, the at least one hinge is a breakable hinge. In some embodiments, the cover and the base are pivotably connected by the at least one hinge.

In some embodiments, the sample substrate rests on top of the base of the substrate holder. In some embodiments, the cover includes a plurality of ribs extending from a surface of the cover, and the gasket is positionally aligned on the surface of the cover by the plurality of ribs. In some embodiments, the gasket is positioned so that when the sample substrate is secured by the substrate holder, and the substrate holder is in a closed position, a vapor-tight seal is formed between the gasket and the sample substrate. In some embodiments, the cover includes at least two apertures.

In some embodiments, the gasket includes at least two openings, wherein the at least two openings are positioned so that when the sample substrate is secured by the substrate holder, and the substrate holder is in a closed position, the at least two apertures are aligned with the at least two openings. In some embodiments, the gasket is configured to prevent fluid transport between the at least two openings when the cover is in the closed position. In some embodiments, the sample substrate is a glass slide. In some embodiments, the base includes a lip extending around a perimeter of the base, the lip configured to retain the sample substrate within the base. In some embodiments, the cover includes a plurality of ribs extending from a surface of the cover, and the sample substrate engages wi th at least one of the plurality of ribs when the substrate holder is in a closed position. In some embodiments, the base includes an opening exposing at least a portion of the sample substrate when the sample substrate is placed in the base.

In some embodiments, each of the first side walls of the cover is generally orthogonal to the second side walls of the cover. In some embodiments, when the substrate holder is in a closed position, the cover is configured to fold onto the base. In some embodiments, the length of the first locking tab is at least 90% of the length of the gasket, e.g., at least 75%, 80%, 90%, 100%, 120%, or at least 150% of the length of the gasket, from about 75% to about 150% of the length of the gasket. In some embodiments, the length of the first locking tab relative to the length of the gasket is about 1:0.9 to about 1: 1.1, about 1:0.8 to about 1: 1.2, about 1:0.5 to about 1: 1.5.

In another aspect, this disclosure is directed to a support device including: a substrate holder including: a cover including opposing first side walls and opposing second side walls, the cover configured to receive a gasket; a base including opposing first side walls and opposing second side walls, the base configured to receive a sample substrate, each of the first side walls of the base and the cover being longer than each of the second side walls of the base and the cover; and a locking tab including a moveable tab and a non-moveable tab, one of the moveable tab and the non-moveable tab coupled to one of the first side walls of the cover and another one of the moveable tab and the non-moveable tab coupled to one of the first side walls of the base, the locking tab having a length about equal to a length of the gasket, wherein the locking tab extends along the length of the gasket.

In some embodiments, the sample substrate rests on top of the base of the substrate holder. In some embodiments, the cover includes a plurality of ribs extending from a surface of the cover, and the gasket is positionally aligned on the surface of the cover by the plurality of ribs. In some embodiments, the gasket is positioned so that when the sample substrate is secured by the substrate holder, and the substrate holder is in a closed position, a vapor-tight seal is formed between the gasket and the sample substrate. In some embodiments, the cover includes at least two apertures. In some embodiments, the gasket includes at least two openings, wherein the at least two openings is positioned so that when the sample substrate is secured by the substrate holder, and the substrate holder is in a closed position, the at least two apertures is aligned with the at least two openings. In some embodiments, the gasket is configured to prevent fluid transport between the at least two openings when the substrate holder is in the closed position.

In some embodiments, the sample substrate includes a glass slide. In some embodiments, the base includes a lip extending around a perimeter of the base, the lip configured to retain the sample substrate within the base. In some embodiments, the cover includes a plurality of ribs extending from a surface of the cover, and the sample substrate engages with at least one of the plurality of ribs when the substrate holder is in a closed position. In some embodiments, the base includes an opening exposing at least a portion of the sample substrate when the sample substrate is placed in the base. In some embodiments, the first side walls of the cover are generally orthogonal to the second side walls of the cover. In some embodiments, the support device further includes at least one breakable hinge extending from one of the second side walls of the cover to one of the second side walls of the base such that, when the substrate holder is in a closed position, the cover is configured to fold onto the base to secure the sample substrate and the gasket between the cover and the base. In some embodiments, the cover and the base are pivotably connected by at least one hinge extending from one of the second side walls of the cover to one of the second side walls of the base such that when the substrate holder is in the closed position, the cover configured to fold over the base to secure the sample substrate and the gasket between the cover and the base. In some embodiments, the cover and the base are pivotably connected by the at least one breakable hinge.

In another aspect, this disclosure is directed to a method of incubating a sample disposed on a sample region of a substrate, the method including: mounting the substrate on a base of a substrate holder of a support device, the substrate including the sample; moving a cover of the substrate holder toward the base of the substrate holder such that first and second moveable tabs on one of the base and the cover engage with first and second non-moveable tabs on the other of the base and the cover, the first moveable tab having a length greater than a length of the second moveable tab and extending along a length of a gasket received by the cover; positioning the substrate and the support device on a heating apparatus; and activating the heating apparatus to transfer heat to the sample.

In some embodiments, the substrate includes a glass slide. In some embodiments, when the substrate holder is coupled to the support device, at least 60% of the sample region is overlaid by the support device. In some embodiments, a height of the first moveable tab is about equal to a height of the gasket. In some embodiments, a length of the first moveable tab is about equal to a length of the gasket.

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.

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.

The term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection, unless expressly stated otherwise, or unless the context of the usage clearly indicates otherwise.

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

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.

FIG. 1 is a perspective view of an example support device in a closed position.

FIG. 2 is a perspective view of the example support device of FIG. 1 in an open position.

FIG. 3 is an exploded view of the example support device of FIG. 1.

FIG. 4 is a perspective view of the example support device of FIG. 1 and a substrate.

FIG. 5 is a perspective view of an example support device in a closed position.

DETAILED DESCRIPTION

I. Introduction This disclosure describes devices and methods 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.

1. Spatial Analysis

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). 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 non-neighboring 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. Differences in analyte levels within different cells in a tissue of a mammal can also provide information on drug resistance mechanisms and the development of the same in a tissue of a mammal. Differences in the presence or absence of analytes within different cells in a tissue of a multicellular organism (e.g., a mammal) can provide information on drug resistance mechanisms and the development of the same in a tissue of a multicellular organism.

The support devices provided herein can be used with spatial analysis methodologies that 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. Spatial analysis methods include, e.g., the use of a capture probe including a spatial barcode (e.g., a nucleic acid sequence that provides information as to the position of the capture probe on a substrate which correlates to a location within a cell or a tissue sample (e.g., mammalian cell or a mammalian tissue sample) and a capture domain that is capable of binding to an analyte (e.g., a protein and/or nucleic acid) produced by and/or present in a cell or a tissue. 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 and correlate a specific spatial barcode with a specific analyte produced by and/or present at a certain location in a cell or tissue (e.g., a mammalian cell or tissue).

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). For example, capture probes can be encapsulated or disposed within a permeable bead (e.g., a gel bead). In some examples, capture probes can be encapsulated within, embedded within, or layered on a surface of a substrate (e.g., any of the exemplary substrates described herein, such as a hydrogel or a porous membrane). When a capture probe is attached to a substrate, for example the surface of a slide, the attachment can be either direct or indirect (e.g., via a linker).

In some examples, a cell or a tissue sample including a cell is contacted with capture probes attached to a substrate (e.g., a surface of a substrate), and the cell or tissue sample is permeabilized to allow analytes to be released from the cell and hybridize to the capture probes attached to the substrate. In some examples, analytes released from the cell passively migrate to the substrate for hybridization to a capture probe (e.g., via gravity). In some examples, analytes released from a cell can be actively directed to the capture probes attached to a substrate using a variety of methods, e.g., electrophoresis, chemical gradient, pressure gradient, fluid flow, or magnetic field.

Non-limiting aspects of support devices are described in PCT/US2019/065100, the entire contents of which are incorporated herein by reference and can be used herein in any combination. Further non-limiting aspects of support devices are described herein.

(a) General Terminology

(i) Biological Samples

The devices and methods described in this disclosure can be used to support substrates configured to receive one or more biological samples. As used herein, “biological sample” is a sample that can be obtained from a subject for analysis using any of a variety of techniques including, but not limited to, biopsy, surgery, and laser capture microscopy

(LCM), and generally includes cells and/or other biological material from the subject, either in suspension or as a tissue section from a tissue sample for example. In addition to the subjects described below, a biological sample can be obtained from non-mammalian organisms (e.g., a plant, an insect, an arachnid, a nematode (e.g., Caenorhabditis elegans), a fungus, an amphibian, or a fish (e.g., zebrafish)). A biological sample can be obtained from a prokaryote such as a bacterium, e.g., Escherichia coli. Staphylococci o Mycoplasma pneumoniae,' an archaeon; a virus such as Hepatitis C virus or human immunodeficiency virus; or a viroid. A biological sample can be obtained from a eukaryote, such as a patient derived organoid (PDO) or patient derived xenograft (PDX). The biological sample can include organoids, a miniaturized and simplified version of an organ produced in vitro in three dimensions that shows realistic micro-anatomy. Organoids can be generated from one or more cells from a tissue, embryonic stem cells, and/or induced pluripotent stem cells, which can self-organize in three-dimensional culture owing to their self-renewal and differentiation capacities. In some embodiments, an organoid is a cerebral organoid, an intestinal organoid, a stomach organoid, a lingual organoid, a thyroid organoid, a thymic organoid, a testicular organoid, a hepatic organoid, a pancreatic organoid, an epithelial organoid, a lung organoid, a kidney organoid, a gastruloid, a cardiac organoid, or a retinal organoid. Subjects from which biological samples can be obtained can be healthy or asymptomatic individuals, individuals that have or are suspected of having a disease (e.g., cancer) or a predisposition to a disease, and/or individuals that are in need of therapy or suspected of needing therapy.

Biological samples can be derived from a homogeneous culture or population of the subjects or organisms mentioned herein or alternatively from a collection of several different organisms, for example, in a community or ecosystem.

Biological samples can include one or more diseased cells. A diseased cell can have altered metabolic properties, gene expression, protein expression, and/or morphologic features. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders, and cancer. Cancer cells can be derived from solid tumors, hematological malignancies, cell lines, or obtained as circulating tumor cells.

Biological samples can also include fetal cells. For example, a procedure such as amniocentesis can be performed to obtain a fetal cell sample from maternal circulation. Sequencing of fetal cells can be used to identify any of a number of genetic disorders, including, e.g., aneuploidy such as Down’s syndrome, Edwards syndrome, and Patau syndrome. Further, cell surface features of fetal cells can be used to identify any of a number of disorders or diseases. Biological samples can also include immune cells. Sequence analysis of the immune repertoire of such cells, including genomic, proteomic, and cell surface features, can provide a wealth of information to facilitate an understanding the status and function of the immune system. By way of example, determining the status (e.g., negative or positive) of minimal residue disease (MRD) in a multiple myeloma (MM) patient following autologous stem cell transplantation is considered a predictor of MRD in the MM patient (see, e.g., U.S. Patent Application Publication No. 2018/0156784, the entire contents of which are incorporated herein by reference).

Examples of immune cells in a biological sample include, but are not limited to, B cells, T cells (e.g., cytotoxic T cells, natural killer T cells, regulatory T cells, and T helper cells), natural killer cells, cytokine induced killer (CIK) cells, myeloid cells, such as granulocytes (basophil granulocytes, eosinophil granulocytes, neutrophil granulocytes/hypersegmented neutrophils), monocytes/macrophages, mast cells, thrombocytes/megakaryocytes, and dendritic cells.

The biological sample can include any number of macromolecules, for example, cellular macromolecules and organelles (e.g., mitochondria and nuclei). The biological sample can be a nucleic acid sample and/or protein sample. The biological sample can be a carbohydrate sample or a lipid sample. The biological sample can be obtained as a tissue sample, such as a tissue section, biopsy, a core biopsy, needle aspirate, or fine needle aspirate. The sample can be a fluid sample, such as a blood sample, urine sample, or saliva sample. The sample can be a skin sample, a colon sample, a cheek swab, a histology sample, a histopathology sample, a plasma or serum sample, a tumor sample, living cells, cultured cells, a clinical sample such as, for example, whole blood or blood-derived products, blood cells, or cultured tissues or cells, including cell suspensions.

Cell-free biological samples can include extracellular polynucleotides. Extracellular polynucleotides can be isolated from a bodily sample, e.g., blood, plasma, serum, urine, saliva, mucosal excretions, sputum, stool, and tears. A biological sample can include a single analyte of interest, or more than one analyte of interest.

(ii) Subject

As used herein, the term “subject” is an animal, such as a mammal (e.g., human or a non-human simian), or avian (e.g., bird), or other organism, such as a plant. Examples of subjects include, but are not limited to, a mammal such as a rodent, mouse, rat, rabbit, guinea pig, ungulate, horse, sheep, pig, goat, cow, cat, dog, primate (i.e. human or non-human primate); a plant such as Arabidopsis thaliana, com, sorghum, oat, wheat, rice, canola, or soybean; an algae such as Chlamydomonas reinhardtii,' a nematode such as Caenorhabditis elegms an insect such as Drosophila melanogaster , mosquito, fruit fly, or honey bee; an arachnid such as a spider; a fish such as zebrafish; a reptile; an amphibian such as a frog or Xenopus laevis,' Dictyostelium discoideunp a fungus such as Pneumocystis carinii, Takifugu rubripes. yeast, Saccharamoyces cerevisiae or Schizosaccharomyces pombe or a Plasmodium falciparum.

(iii) Substrate Attachment

In some embodiments, the biological sample can be attached to a substrate. Examples of substrates suitable for this purpose are described in detail below. Attachment of the biological sample can be irreversible or reversible, depending upon the nature of the sample and subsequent steps in the analytical method.

In certain embodiments, the sample can be attached to the substrate reversibly by applying a suitable polymer coating to the substrate and contacting the sample to the polymer coating. The sample can then be detached from the substrate using an organic solvent that at least partially dissolves the polymer coating. Hydrogels are examples of polymers that are suitable for this purpose.

More generally, in some embodiments, the substrate can be coated or functionalized with one or more substances to facilitate attachment of the sample to the substrate. Suitable substances that can be used to coat or functionalize the substrate include, but are not limited to, lectins, poly-lysine, antibodies, and polysaccharides.

(iv) Substrates

For analytical methods using a substrate (e.g., spatial array-based analytical methods), the substrate functions as a support for direct or indirect attachment of capture probes to features of the array. In addition, in some embodiments, a substrate (e.g., the same substrate or a different substrate) can be used to provide support to a biological sample, particularly, for example, a thin tissue section. Accordingly, as used herein, a “substrate” is a support that is insoluble in aqueous liquid and which allows for positioning of biological samples, analytes, features, and/or capture probes on the substrate.

Further, a “substrate” as used herein, and when not preceded by the modifier “chemical,” refers to a member with at least one surface that generally functions to provide physical support for biological samples, analytes, and/or any of the other chemical and/or physical moieties, agents, and structures that can be used with various analytical methods. Substrates can be formed from a variety of solid materials, gel-based materials, colloidal materials, semi-solid materials (e.g., materials that are at least partially cross-linked), materials that are fully or partially cured, and materials that undergo a phase change or transition to provide physical support. Examples of substrates that can be used in the methods and devices described herein include, but are not limited to, slides (e.g., slides formed from various glasses, slides formed from various polymers), hydrogels, layers and/or films, membranes (e.g., porous membranes), wafers, plates, or combinations thereof. In some embodiments, substrates can optionally include functional elements such as recesses, protruding structures, microfluidic elements (e.g., channels, reservoirs, electrodes, valves, seals), and various markings, as will be discussed in further detail below.

1) Substrate Attnbutes

A substrate can generally have any suitable form or format that can be accommodated by the device disclosed herein. For example, a substrate can be flat, curved, e.g., convexly or concavely curved towards the area where the interaction between a biological sample, e.g., tissue sample, and a substrate takes place. In some embodiments, a substrate is flat, e.g., planar, chip, or slide. A substrate can contain one or more patterned surfaces within the substrate (e.g., channels, wells, projections, ridges, divots, etc.).

A substrate can be of any desired shape. For example, a substrate can be typically a thin, flat shape (e.g., a square or a rectangle). In some embodiments, a substrate structure has rounded comers (e.g., for increased safety or robustness). In some embodiments, a substrate structure has one or more cut-off comers (e.g., for use with a slide clamp or cross-table). In some embodiments, where a substrate structure is flat, the substrate stmcture can be any appropriate type of support having a flat surface (e.g., a chip or a slide such as a microscope slide).

Substrates can optionally include various structures such as, but not limited to, projections, ridges, and channels. A substrate can be micropattemed to limit lateral diffusion (e.g., to prevent overlap of spatial barcodes). A substrate modified with such structures can be modified to allow association of analytes, features (e.g., specific locations where barcodes are deposited, beads), or probes at individual sites. For example, the sites where a substrate is modified with various structures can be contiguous (e.g., the sites can be located within an area of the substrate that is enclosed by one of the gasket openings when the device is in a closed position) or non-contiguous with other sites (e.g., a first site can be located within a first area of the substrate that is enclosed by a first gasket opening and a second site can be located within a second area of the substrate that is enclosed by a second gasket opening when the support device is in a closed position). In some embodiments, the surface of a substrate can be modified so that discrete sites are formed that can only have or accommodate a single feature. In some embodiments, the surface of a substrate can be modified so that features are located at random sites (e.g., random sites within an area of the substrate that is enclosed by a gasket opening when the support device is in a closed position).

In some embodiments, the surface of a substrate is modified to contain one or more wells, using techniques such as (but not limited to) stamping, microetching, or molding techniques. In some embodiments in which a substrate includes one or more wells, the substrate can be a concavity slide or cavity slide. For example, wells can be formed by one or more shallow depressions on the surface of the substrate. In some embodiments, where a substrate includes one or more wells, the wells can be formed by attaching a cassette (e.g., a cassette containing one or more chambers) to a surface of the substrate structure.

In some embodiments, the structures of a substrate (e.g., wells or features) can each bear one or more different capture probes. Different capture probes attached to each structure can be identified according to the locations of the structures in or on the surface of the substrate. Exemplary substrates include arrays in which separate structures are located on the substrate including, for example, those having wells or locations on the substate that accommodate features.

In some embodiments where the substrate is modified to contain one or more structures, including but not limited to, wells, projections, ridges, features, or markings, the structures can include physically altered sites. For example, a substrate modified with various structures can include physical properties, including, but not limited to, physical configurations, magnetic or compressive forces, chemically functionalized sites, chemically altered sites, and/or electrostatically altered sites. In some embodiments where the substrate is modified to contain various structures, including but not limited to wells, projections, ridges, features, or markings, the structures are applied in a pattern. Alternatively, the structures can be randomly distributed.

The substrate (e g., or a bead or a feature on an array) can include tens to hundreds of thousands or millions of individual oligonucleotide molecules (e.g., at least about 10,000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 100,000,000, 1,000,000,000, or 10,000,000,000 oligonucleotide molecules).

In some embodiments, a substrate includes one or more markings on a surface of a substrate, e.g., to provide guidance for correlating spatial information with the characterization of the analyte of interest. For example, a substrate can be marked with a grid of lines (e.g., to allow the size of objects seen under magnification to be easily eshmated and/or to provide reference areas for counting objects). In some embodiments, fiducial markers can be included on a substrate. Such markings can be made using techniques including, but not limited to, printing, sandblasting, and depositing on the surface.

A wide variety of different substrates can be used for the foregoing purposes. In general, a substrate can be any suitable support material that can be accommodated by the disclosed device. Exemplary substrates include, but are not limited to, glass, modified and/or functionalized glass, hydrogels, films, membranes, plastics (including e.g., acrylics, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™, cyclic olefins, polyimides etc.), nylon, ceramics, resins, Zeonor, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, optical fiber bundles, and polymers, such as polystyrene, cyclic olefin copolymers (COCs), cyclic olefin polymers (COPs), polypropylene, polyethylene polycarbonate, or combinations thereof.

Among the examples of substrate materials discussed above, polystyrene is a hydrophobic material suitable for binding negatively charged macromolecules because it normally contains few hydrophilic groups. For nucleic acids immobilized on glass slides, by increasing the hydrophobicity of the glass surface the nucleic acid immobilization can be increased. Such an enhancement can permit a relatively more densely packed formation (e.g., provide improved specificity and resolution).

2) Conductive Substrates

In some embodiments, the substrate can be a conductive substrate. Conductive substrates (e.g., electrophoretic compatible arrays) generated as described herein can be used in the spatial detection of analytes.

In some embodiments, a conductive substrate can include glass (e.g., a glass slide) that has been coated with a substance or otherwise modified to confer conductive properties to the glass. In some embodiments, a glass slide can be coated with a conductive coating. In some embodiments, a conductive coating includes tin oxide (TO) or indium tin oxide (ITO). In some embodiments, a conductive coating includes a transparent conductive oxide (TOO). In some embodiments, a conductive coating includes aluminum doped zinc oxide (AZO). In some embodiments, a conductive coating includes fluorine doped tin oxide (FTO).

In some embodiments, arrays that are spotted or printed with oligonucleotides (e.g., capture probes) can be generated on a conductive substrate (e.g., any of the conductive substrates described herein). For example, the arrays described herein can be compatible with active analyte capture methods (e.g., including without limitation, electrophoretic capture methods). In some embodiments, a conductive substrate is a porous medium. Non-limiting examples of porous media that can be used in methods that employ active analyte capture include a nitrocellulose or nylon membrane. In some embodiments, a porous medium that can be used in methods described herein that employ active analyte capture includes paper. In some embodiments, the oligonucleotides can be printed on a paper substrate. In some embodiments, the printed oligonucleotides can interact with the substrate (e.g., interact with fibers of the paper). In some embodiments, printed oligonucleotides can covalently bind the substrate (e.g., to fibers of the paper). In some embodiments, oligonucleotides in a molecular precursor solution can be printed on a conductive substrate (e.g., paper). In some embodiments, a molecular precursor solution can polymerize, thereby generating gel pads on the conductive substrate (e.g., paper). In some embodiments, a molecular precursor solution can be polymerized by light (e.g., photocured). In some embodiments, gel beads containing oligonucleotides (e.g., barcoded oligonucleotides such as capture probes) can be printed on a conductive substrate (e.g., paper). In some embodiments, the printed oligonucleotides can be covalently attached into the gel matrix.

3) Coatings

In some embodiments, a surface of a substrate can be coated with a cell-permissive coating to allow adherence of live cells. A “cell-permissive coating” is a coating that allows or helps cells to maintain cell viability (e.g., remain viable) on the substrate. For example, a cell-permissive coating can enhance cell attachment, cell grow th, and/or cell differentiation, e.g., a cell-permissive coating can provide nutrients to the live cells. A cell-permissive coating can include a biological material and/or a synthetic material. Non-limiting examples of a cell-permissive coating include coatings that feature one or more extracellular matrix (ECM) components (e.g., proteoglycans and fibrous proteins such as collagen, elastin, fibronectin and laminin), poly-lysine, poly(L)-omithine, and/or a biocompatible silicone (e.g., CYTOSOFT®). For example, a cell-permissive coating that includes one or more extracellular matrix components can include collagen Type I, collagen Type II, collagen Type IV, elastin, fibronectin, laminin, and/or vitronectin. In some embodiments, the cell- permissive coating includes a solubilized basement membrane preparation extracted from the Engelbreth-Holm- Swarm (EHS) mouse sarcoma (e.g., MATRIGEL®). In some embodiments, the cell-permissive coating includes collagen. A cell-permissive coating can be used to culture adherent cells on a spatially-barcoded array, or to maintain cell viability of a tissue sample or section while in contact with a spatially -barcoded array.

In some embodiments, a substrate is coated with a surface treatment such as poly(L)- lysine. Additionally or alternatively, the substrate can be treated by silanation, e.g., with epoxy -silane, amino-silane, and/or by a treatment with polyacrylamide.

In some embodiments, a substrate is treated in order to minimize or reduce nonspecific analyte hybridization within or between features. For example, treatment can include coating the substrate with a hydrogel, film, and/or membrane that creates a physical barrier to non-specific hybridization. Any suitable hydrogel can be used. For example, hydrogel matrices prepared according to the methods set forth in U.S. Patent Nos. 6,391,937, 9,512,422, and 9,889,422, and U.S. Patent Application Publication Nos. U.S. 2017/0253918 and U.S. 2018/0052081, can be used. The entire contents of each of the foregoing documents is incorporated herein by reference.

Treatment can include adding a functional group that is reactive or capable of being activated such that it becomes reactive after application of a stimulus (e.g., photoreactive functional groups). Treatment can include treating with polymers having one or more physical properties (e.g., mechanical, electrical, magnetic, and/or thermal) that minimize nonspecific binding (e.g., that activate a substrate at certain locations to allow analyte hybridization at those locations).

A “removeable coating” is a coating that can be removed from the surface of a substrate upon application of a releasing agent. In some embodiments, a removeable coating includes a hydrogel as described herein, e.g., a hydrogel including a polypeptide-based material. Non-limiting examples of a hydrogel featuring a polypeptide-based material include a synthetic peptide-based material featuring a combination of spider silk and a transmembrane segment of human muscle L-type calcium channel (e.g., PEPGEL®), an amphiphilic 16 residue peptide containing a repeating arginine-alanine-aspartate-alanine sequence (RAD ARAD ARAD ARADA) (e g., PURAMATRIX®), EAK16 (AEAEAKAKAEAEAKAK), KLD12 (KLDLKLDLKLDL), and PGMATRIX™.

In some embodiments, the hydrogel in the removeable coating is a stimulus- responsive hydrogel. A stimulus-responsive hydrogel can undergo a gel-to-solution and/or gel-to-solid transition upon application of one or more external triggers (e.g., a releasing agent). See, e.g., Willner, Acc. Chem. Res. 50:657-658, 2017, which is incorporated herein by reference in its entirety. Non-limiting examples of a stimulus-responsive hydrogel include a thermoresponsive hydrogel, a pH-responsive hydrogel, a light-responsive hydrogel, a redox-responsive hydrogel, an analyte-responsive hydrogel, or a combination thereof. In some embodiments, a stimulus-responsive hydrogel can be a multi-stimuli-responsive hydrogel.

A “releasing agent” or “external trigger” is an agent that allows for the removal of a removeable coating from a substrate when the releasing agent is applied to the removeable coating. An external trigger or releasing agent can include physical triggers such as thermal, magnetic, ultrasonic, electrochemical, and/or light stimuli as well as chemical triggers such as pH, redox reactions, supramolecular complexes, and/or biocatalytically driven reactions. See e.g., Echeverria, et al., Gels (2018), 4, 54; doi: 10.3390/gels4020054, which is incorporated herein by reference in its entirety. The type of “releasing agent” or “external trigger” can depend on the type of removeable coating. For example, a removeable coating featuring a redox-responsive hydrogel can be removed upon application of a releasing agent that includes a reducing agent such as dithiothreitol (DTT). As another example, a pH-responsive hydrogel can be removed upon the application of a releasing agent that changes the pH. In some embodiments, the biological sample can be confined to a specific region or area. For example, a biological sample can be affixed to a glass slide and a chamber, gasket, or cage positioned over the biological sample to act as a containment region or frame within which the biological sample is deposited.

SEQ ID NO: 7 GGTGACTCTAGATAACCT

2. Additional Support Device Embodiments

In some embodiments, a support device can be part of a system (e.g., a system 3102, as described in PCT/US2019/065100, the entire contents of which are incorporated herein by reference) for heating a substrate that can further include a plate. The plate can be configured to be received by a heating device (e.g., a thermocycler) and provide heat transfer between the heating device and the support device. The support device (e.g., a substrate holder 3150, as described in PCT/US2019/065100) can hold one or more substrates (such as one or more glass slides) and can removably couple to the plate to facilitate heat transfer from the plate to the one or more substrates. The support device can include a bottom member and a top member. In some embodiments, the substrate device can further include a slide. In some embodiments, the support device can include a gasket that is positioned inside the support device. In some embodiments, the support device can include an engagement mechanism (e.g., screws) for coupling the bottom member and the top member.

In some embodiments, the support device can be a single-piece component (e.g., substrate holder 4400, as described in PCT/US2019/065100) that receives a gasket and a substrate. In some embodiments, the support device can include one or more fasteners (such as a side mounted press latch 4410, as described in PCT/US2019/065100) for snap engagement of a substate. In some embodiments, the support device can further include one or more tabs (e.g., first tab 4412a and second tab 4412b, as described in PCT/US2019/065100) that are configured to engage the substrate. The support device can include a bottom surface defining a plurality of apertures that are configured to align with a plurality of apertures of a gasket.

In some embodiments, the support device can include a top component and a bottom component that are connected via one or more hinges (e.g., hinge 7360, as described in PCT/US2019/065100) extending from a side wall of the bottom component. The support device can further include one or more engagement features protruding from a side wall of the top component (e.g., first notch 7358a and second notch 7358b, as described in PCT/US2019/06510), which are configured to engage one or more tabs protruding from a side wall of the bottom component, thereby enabling closure of the support device.

II. Support Devices

Embodiments may provide one or more of the following advantages.

The devices provided herein can be used to ensure that the temperature of a substrate and any samples and/or reagents supported on the substrate surface is controlled uniformly and consistently during a sample preparation and/or analysis protocol. During such protocols, uneven heating can lead to failure of the sample preparation. Further, even when sample heating is relatively uniform, condensation that contacts the sample may impair certain reactions that are part of the protocol, or otherwise affect the chemical reactions that occur. The devices described, in various embodiments, provide for heating of multiple surfaces of a substrate (e.g., in a slide cassette or substrate holder), and can include features that facilitate heat transfer from heating elements to the substrate, and that can reduce or prevent condensation from forming in certain regions of the substrate (e.g., in sample wells or regions on the substrate surface). Additionally, the devices provided herein can provide mitigation of cross-contamination of samples and/or reagents from different locations on the substrate. For example, the described gaskets of the device can provide for discrete biological sample areas and a vapor barrier from one well to the next. Further, the gaskets can impede reagent spillage or flow from one well to the next. In some embodiments, the devices provided herein include a locking that helps secure the gasket in place when the support device is in a closed position.

Referring generally to FIGS. 1-4, an embodiment of an example support device can include a substrate and a substrate holder. The substrate holder can include a gasket, a cover, and a base. In some embodiments, the cover and the base are integrally connected (e.g., the substrate holder is a one-part design). In some embodiments, the substrate holder is manufactured using injection molding techniques. Non-limiting materials used to manufacture the support devices of the disclosure include polypropylene homopolymers. In some embodiments, the substrate holder is disposable. In some embodiments, the substrate holder is reusable.

In some embodiments, the substrate holder receives a substrate, such as a slide, for example a glass slide. In some embodiments, the substrate holder includes an attachment mechanism to couple and/or secure the substrate to the substrate holder. The cover can be configured to receive a gasket or can be co-molded with a gasket. The cover can include a plurality of ribs extending from a surface of the cover. Furthermore, the base can be configured to receive a substrate. In some examples, the support device can further include at least one pair of locking tabs where each locking tab includes a moveable tab that is coupled to a first side wall of the cover and a non-moveable tab coupled to a first side wall of the base. In other examples, each locking tab includes a moveable tab that is coupled to a first side wall of the base and a non-moveable tab is coupled to a first side wall of the cover. The moveable tab can be configured to engage with the non-moveable tab to releasably secure the cover to the base. In some embodiments, a first locking tab of the pair of locking tabs has a length that is greater than the length of a second locking tab of the pair of locking tabs.

In some examples, the cover and the base are pivotably connected together by at least one hinge. In some embodiments, the hinge can be a living hinge. In some embodiments, the cover and the base are pivotably connected together by two or more hinges. In some embodiments, at least one hinge extends from a second side wall of the cover to a second side wall of the base. For example, when the substrate holder is in a closed position, the cover can be configured to fold onto the base to secure the substrate and the gasket between the cover and the base. In some embodiments, the pivotable connection can be broken at any stage, such that there is no longer a pivotable connection between the cover and the base.

Moreover, the support device can include a substrate (e.g., a glass slide) that is configured to receive a sample. In some embodiments, the sample can be a biological sample. In some embodiments, the sample can be any of the biological samples defined elsewhere in the disclosure. The substrate includes a first surface and a second surface. In some examples, the first surface of the substrate is configured to receive a sample. In some embodiments, the substrate includes a sample region. In some examples, the sample region receives one or more samples.

FIG. 1 shows support device 100 in a closed position. Support device 100 includes a substrate holder 101 and a substrate 110. In particular, FIG. 1 shows the substrate holder 101 having a base 134 and a cover 136. Base 134 and cover 136 have a first pair of side walls 103 and a second pair of side walls 105. Each of the first side walls 103 can be longer than each of the second side walls 105. Thus, base 134 and cover 136 have a substantially rectangular shape. In some embodiments, base 134 and cover 136 have a circular, square, triangular, or any other suitable shape. In some embodiments, the first side wall 103 of the cover 136 is generally orthogonal to the second side wall 105 of the cover 136. For example, the first side wall 103 and the second side wall 105 of the cover 136 can form an angle between 85 and 95 degrees. In some embodiments, the first side wall 103 of the base 134 is generally orthogonal to the second side wall 105 of the base 134. For example, the first side wall 103 and the second side wall 105 of the base 134 can form an angle between 85 and 95 degrees. The cover 136 includes a top surface 102 where a plurality of apertures 104 are defined through. In some embodiments, the plurality of apertures 104 can be defined such that it aligns with a plurality of openings of a gasket when the gasket is positioned between the cover 136 and the base 134 in a closed position. In some embodiments, the plurality of apertures 104 can be defined such that it aligns with a sample region of the substrate (e.g., a slide) when the support device is in a closed position. In some embodiments, the plurality of apertures 104 can provide a user with access to the substrate (e g., a sample region on the substrate) to view a sample on the sample region and/or deliver a solution directly onto the sample on the sample region or onto a surface of the substrate, for example, when the support device is in a closed position.

The substrate holder 101 can include at least one pair of locking tabs 107 that are configured to releasably secure, close, lock, fasten, and/or engage the base 134 with the cover 136. Each locking tab 107 can include a moveable tab 106 coupled to a first side wall 103 of the cover 136 and a non-moveable tab 108 coupled to a first side wall 103 of the base 134. In some embodiments, the moveable tab 106 is configured to engage with the non-moveable tab 108 to releasably secure the cover 136 to the base 134. In alternative examples, moveable tab 106 may be coupled to a second side wall 105 of the cover 136 and a non-moveable tab 108 may be coupled to a second side wall 105 of the base 134. In some embodiments, one or more locking tabs 107 may be coupled to one or both first side walls 103 and/or one or more locking tabs 107 may be coupled to one or both second side walls 105. In some embodiments, the moveable tabs 106 and the cover 136 are integrally joined. For example, the moveable tabs 106 and the cover 136 can be formed as a single component in a molding, e.g., injection molding, process. In some embodiments, the non-moveable tabs 108 and the base 134 are integrally joined. For example, the non-moveable tabs 108 and the base 134 can be formed as a single component in a molding, e.g., injection molding, process. In some examples, the substrate holder 101 can include multiple locking tabs 107 (e.g., at least 2, 3, 4, or 5 pairs of locking tabs 107). In some embodiments, the substrate holder 101 includes one locking tab. In some embodiments, any type of fastener that allows releasable engagement of the base 134 with the cover 136 can be used, such as, for example, magnetic fasteners, snap-fits, hook- and-loop fasteners, press latches, screws, press fit type connectors (e.g., lever, a clip, or a clamp), or any combination thereof. In some embodiments, the substrate holder 101 can further include one or more spring-loaded fasteners.

The substrate holder 101 can include a locking tab 158 that is configured to releasably secure, close, lock, fasten, and/or engage the base 134 with the cover 136. The locking tab 158 can include a moveable tab 162 coupled to a first side wall 103 of the cover 136 and a non-moveable tab 160 coupled to a first side wall 103 of the base 134. In some embodiments, the moveable tab 162 is configured to engage with the non-moveable tab 160 to releasably secure the cover 136 to the base 134. In alternative examples, the moveable tab 162 may be coupled to a second side wall 105 of the cover 136 and a non-moveable tab 160 may be coupled to a second side wall 105 of the base 134. In some embodiments, one or more locking tabs 158 may be coupled to one or both first side walls 103 and/or one or more locking tabs 158 may be coupled to one or both second side walls 105. In some embodiments, the moveable tab 162 and the cover 136 are integrally joined. In some embodiments, the non- moveable tabs 160 and the base 134 are integrally joined. In some examples, substrate holder 101 can include multiple locking tabs 158 (e.g., at least 2, 3, 4, or 5 locking tabs 158). In some embodiments, the substrate holder 101 includes one locking tab 158 and one locking tab 107. In some embodiments, the substrate holder 101 includes one locking tab 158 and a pair of locking tabs 107. In some embodiments, the substrate holder 101 includes one locking tab 158 and three locking tabs 107. In some embodiments, the substrate holder 101 includes one locking tab 158 and four locking tabs 107.

As shown in FIG. 1, a surface of substrate 110 is exposed through a plurality of apertures 104 of the substrate holder 101. The plurality of apertures 104 are defined by the top surface 102 of cover 136. The plurality of apertures 104 align with the plurality of openings of the gasket when the support device 100 is in the closed position.

FIG. 2 is a perspective view of the substrate holder 101 of FIG. 1 in an open position. The substrate holder 201 includes the locking tab 258 including a moveable tab 262 having a length Z_m and a non-moveable tab 260 having a length In. The substrate holder 201 includes the pair of locking tabs 207 each including a moveable tab 206 having a length / ’_m and a non- moveable tab 208 having a length / ’_n. A length of the locking tab 258 can be greater than a length of the pair of locking tabs 207. For example, the length Z_m of the moveable tab 262 can be greater than the length / ’_m of the moveable tab 206. In some embodiments, the length Z_m of the moveable tab 262 can be about 40% greater than the length / ’_m of the moveable tab 206. In some embodiments, the length Zm of the moveable tab 262 can be at least about 10% to about 80% (e.g., between about 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, or 70% to 80%) greater than the length Z ’_m of the moveable tab 206. The length Zn of the non-moveable tab 260 can be greater than the length Z ’_n of the non-moveable tab 208. In some embodiments, the length Z_n of the non-moveable tab 260 can be about 40% greater than the length Z’n of the non-moveable tab 208. In some embodiments, the length Z_n of the moveable tab 260 can be at least about 10% to about 80% (e.g., between about 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, or 70% to 80%) or more greater than the length Z’n of the moveable tab 208.

In some embodiments, the length Z_m of the moveable tab 262 is about equal to the length Z_g of the gasket 224 or the length Z_g of the gasket 224. In some embodiments, the length Zm of the moveable tab 262 can be about 5% greater than the length Z_g of the gasket 224. In some embodiments, the length Zm of the moveable tab 262 can be at least about 5% to about 20% (e.g., between about 5% to 10%, 10% to 15%, 15% to 20%) greater than the length Z_g of the gasket 224. In some embodiments, the length Z_m of the moveable tab 262 is aligned with a length Z_g of the gasket 224. In some embodiments, the length Z of the moveable tab 262 is at least 90% of the length Z_g of the gasket 224, e.g., at least 75%, 80%, 90%, 100%, 120%, or at least 150% of the length Z_g of the gasket 224, from about 75% to about 150% of the length Z_g of the gasket 224. In some embodiments, the length Z_m of the moveable tab 262relative to the length Z_g of the gasket 224 is about 1 :0.9 to about 1: 1.1, about 1 :0.8 to about 1: 1.2, about 1:0.5 to about 1 : 1.5. The length Z_m of the moveable tab 262 can help secure the gasket 224 when substrate holder 201 is in a closed position such that a force is exerted onto gasket wall 209, along the length Z_g of the gasket 224. In some embodiments, moveable tab 262 is centered about the gasket 224 (e.g., centered along the length Z_mof gasket 224). The relatively larger length of the locking tab 258 compared to the length of the locking tab 207 can allow the force exerted onto the gasket wall 209 to be more easily distributed across an entire length of the gasket 224. In addition, the relatively larger length of the locking tab 258 can allow a user to more easily manipulate the locking tab 258 (e.g., for locking or releasing the locking tab 258).

In FIG. 2, the cover 236 and base 234 are pivotably connected by a pair of hinges 216 that extend from a second side wall 205 of the cover 236 to a second side wall 205 of the base 234. In some embodiments, there are no hinges. In some embodiments, the pair of hinges can be a pair of living hinges. In some embodiments, the cover and base are pivotably connected by at least one hinge. In some embodiments, one or more hinges can extend from a second side wall of the cover to a second side wall of the base. In some embodiments, the cover and the base can be pivotably connected by one hinge that has a length spanning about 50% or more of the length of the side from which it extends from. In some embodiments, the cover and the base are pivotably connected by several hinges. In some embodiments, if there are hinges present the hinges are breakable, such that the cover and the base are separable upon breaking the one or more hinges.

FIG. 2 shows the substrate holder 201 in an open position, having the cover 236 in an extended, unfolded position away from the base 234. In some embodiments, the substrate holder 201 is in an open position when the cover 236 is at about 180° with respect to the base 234, as shown in FIG. 2. In some embodiments, the substrate holder 201 is in an open position when the cover 236 is at an angle ranging from about 10° to about 170° with respect to the base 234.

The cover 236 has a bottom surface 232 from which a plurality of ribs 218 can extend perpendicularly from. In some embodiments, the cover 236 is configured to receive the gasket 224. In some embodiments, a gasket 224 can withstand at least about 10% compression set. In some embodiments, the gasket 224 can withstand about 15% or more compression set. In some embodiments, the gasket 224 can withstand about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 40%, 60%, 70% or more compression set. As used herein, compression set is expressed as the percentage of the original specimen thickness after being exposed to a constant compressive force. In some embodiments, the gasket 224 is co-molded with a portion of the substrate holder such as, for example, the cover 236. In some embodiments, the gasket 224 can withstand a compression force that results in a gasket height hg change of about 0.5 millimeters (mm). In some embodiments, the gasket 224 can withstand a compression force that results in a gasket height h_& change of about 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm or more. In some embodiments, the gasket 224 is constructed out of a thermoplastic polymer, such as a thermoplastic elastomer. In some embodiments, the gasket 224 removably adheres to the bottom surface 232 of the cover 236.

The gasket 224 can include a plurality of openings 238. In some embodiments, the plurality of openings 238 can align with the plurality of apertures 104 defined by the surface of the cover 236. For example, in some embodiments, the plurality of openings 238 is positioned so that when the substrate 110 (shown in FIG. 1) rests on top of the substrate holder 201, and the substrate holder 201 is in a closed position, the plurality of apertures 104 are aligned with the plurality of openings 238. In some embodiments, the gasket 224 applies pressure on the substrate 210 when substrate holder 201 is in a closed position. The plurality of ribs 218 can be configured to support the substrate 210 and the gasket 224. In some embodiments, the gasket 224 is positionally aligned on the surface of the cover 236 by the plurality of ribs. In some embodiments, the gasket 224 is positioned so that when the substrate 210 rests on top of the substrate holder 201, and the substrate holder 201 is in the closed position, a vapor-tight seal is formed between the gasket 224 and the substrate 210. In some embodiments, an air-tight seal is formed between the gasket 224 and the substrate 210. In some embodiments, the gasket 224 is configured to prevent fluid transport between the plurality of openings 238 when the cover 236 is in the closed position.

Moveable tab 262 includes a top portion 268 extending from a substantially vertical wall 270 that further extends from the cover 236. The vertical wall 270 engages with the main body of the non-moveable tab 260 (e.g., its mating part) when the substrate holder 201 is in a closed position. The top portion 268 extends outwardly away from the main body of moveable tab 262 to form a gripping handle for the user. In some embodiments, the gripping handle may facilitate pulling or pushing of moveable tab 262 by the user (e.g., when engaging and/or disengaging non-moveable tab 260). The top portion 268 extends outwardly away from the main body of moveable tab 262 at an angle a, with respect to a horizontal plane that is parallel to the bottom surface 232 of cover 236. In some embodiments, angle a can be about 15 degrees. In some embodiments, angle a can be at least about 5 degrees to about 90 degrees or more (e.g., between about 5 degrees to 10 degrees, 10 degrees to 15 degrees, 15 degrees to 20 degrees, 20 degrees to 25 degrees, 25 degrees to 30 degrees, 30 degrees to 35 degrees, 35 degrees to 40 degrees, 40 degrees to 45 degrees, 45 degrees to 50 degrees, 50 degrees to 55 degrees, 55 degrees to 60 degrees, 60 degrees to 65 degrees, 65 degrees to 70 degrees, 70 degrees to 75 degrees, 75 degrees to 80 degrees, 80 degrees to 85 degrees, 85 degrees to 90).

A first height h_m includes a top portion 268 and vertical wall 270 of moveable tab 262. A second height hv includes the vertical wall 270 of moveable tab 262. In some embodiments, a first height h of the moveable tab 262 is about equal to the height hg of the gasket 224. In some embodiments, the first height h of the moveable tab 262 can be about 5% greater than the height hg of the gasket 224. In some embodiments, the first height hm of the moveable tab 262 can be at least about 5% to about 20% (e.g., between about 5% and 10%, 10% and 15%, 15% and 20%) greater than the height hg of the gasket 224. The first height hm of the moveable tab 262 can help secure the gasket 224 when substrate holder 201 is in a closed position such that a force is exerted onto the gasket wall 209 adjacent to the locking tab 258.

In some embodiments, a second height h of the moveable tab 262 can be about equal to the height hg of the gasket 224. In some embodiments, the second height h_v of the moveable tab 262 can be about 5% greater than the height hg of the gasket 224. In some embodiments, the second height h? of the moveable tab 262 can be at least about 5% to about 20% (e.g., between about 5% and 10%, 10% and 15%, 15% and20%) greater than the height hg of the gasket 224. The second height h_v of the moveable tab 262 can help secure the gasket 224 when substrate holder 201 is in a closed position such that a force is exerted onto the gasket wall 209 adjacent to the locking tab 258.

In the example shown in FIG. 2, the moveable tabs 206 each include a corresponding opening 281, and the moveable tab 262 includes an opening 282. The openings 281 and the opening 282 are vertically extended openings within which the non-moveable tabs 208, 260 engage with the moveable tabs 206 in the closed position of the substrate holder 201. The non-moveable tabs 208, 260 protrude horizontally outwardly from side walls of the base 234 (e.g., the first side wall 103 shown in FIG. 1). Because the non-moveable tabs 208, 260 protrude horizontally outward, the non-moveable tabs 208, 260 contact portions of the moveable tabs 206, 262 as the substrate holder 201 is moved from the open position (FIG. 2) to the closed position (FIG. 1).

For example, the non-moveable tabs 208 contact sloped portions 271 of the moveable tabs 206 and the non-moveable tabs 260 contact a sloped portion 272 of the moveable tab 262 as the cover 236 and the base 234 are brought toward one another to move the substrate holder 201 from the open position (FIG. 2) to the closed position (FIG. 1). This contact causes the moveable tabs 206, 262 to deflect outwardly, allowing the non-moveable tabs 208, 260 to move beyond the sloped portions 271, 272 of the moveable tabs 206, 262 into the openings 281, 282. In some embodiments, to facilitate deflection of the moveable tab 262, the user can operate the gripping handle of the moveable tab 262. Once the non-moveable tabs 208, 260 move beyond the sloped portions 271, 272 into the openings 281, 282, the moveable tabs 206, 262 return toward their neutral positions such that surfaces of the moveable tabs 206, 262 defining the openings 281, 282 are configured to contact the horizontally protruding portions of the non-moveable tabs 206, 262. This creates a locking engagement between the non-moveable tabs 208, 260 and the moveable tabs 206, 262 that prevents the base 234 and the cover 236 from being moved away from one another. When the non-moveable tabs 208, 260 are positioned within the openings 281, 282, the substrate holder 201 is in the closed position. In the closed position of the substrate holder, the gasket 224 contacts (e.g., is compressed against) the substrate and provides a fluid tight seal between the sample regions of the substrate. In the example shown in FIG. 2, the substrate holder 201 includes a plurality of ribs extending perpendicularly or protruding outwardly from bottom surface 232. The gasket 224 does not come in contact (i.e., does not abut) the plurality of ribs 218. In some embodiments, the gasket is not co-molded with the cover. The gasket is mounted to the cover in another appropriate manner. In such embodiments, one or more ribs can be in proximity to or abut one or more gasket walls to help retain the gasket in a proper position. In some embodiments, when the gasket is not co-molded with the cover, one or more ribs can be in proximity to or abut 1, 2, 3, or 4 of the gasket walls. In some embodiments, the plurality of ribs 218 frame an area of the bottom surface 232 (e.g., an area that is sufficiently sized and configured to receive the gasket 224). The plurality of ribs 218 can be disposed parallel to the second side walls 205. In some embodiments, the gasket is co-molded with the cover.

The plurality of ribs 218 has a width w. In some embodiments, the plurality of ribs 218 can have an equal width w, as shown in FIG. 2. In some embodiments, the widths of the plurality of ribs 218 can vary. One of ribs 218 extends substantially perpendicular from bottom surface 232 near a first end 21 la of substrate holder 201. The remaining ribs 218 extend perpendicular from bottom surface 232 near a second end 211b of substrate holder 201. In some embodiments, the substrate holder 201 may include multiple ribs (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more ribs). In some embodiments, the substrate holder 201 can include one or more ribs that extend perpendicular to the plurality of ribs 218.

The plurality of ribs 218 has a height h. In some embodiments, the plurality of nbs 218 can have an equal height h, as shown in FIG. 2. In some embodiments, a height h can be provided such that the top edge of the plurality of ribs 218 comes in contact with substrate 110 (shown in FIG. 1) when the support device is in the closed position (FIG. 1). In embodiments in which the gasket 224 is compressed against the substrate 110, the contact between the plurality of ribs 218 and the substrate 110 can define a maximum amount of compression of the gasket 224. In particular, further compression of the gasket 224 can be prevented when the top edge of the plurality of ribs 218 contacts the substrate 110. In some embodiments, the plurality of ribs 218 secures substrate 110 by coming in contact with a surface of substrate 110 when the support device is in the closed position (FIG. 1). In some embodiments, the first surface 228 of the substrate 110 engages with at least one of the plurality of ribs 218 when the substrate holder 101 is in the closed position (FIG. 1). In some embodiments, the first surface 228 of the substrate 110 includes a sample region. In some embodiments, the sample region is an area of the substrate that is configured to receive one or more samples. In some embodiments, substrate 110 includes one or more sample regions.

FIG. 3 is an exploded view of an exemplary support device 300 in an open position. Cover 336 is configured to receive gasket 324. The cover 336 defines four holes 342 that receive four feet 344 of gasket 324. Each foot 344 projects from a bottom surface 366 of the gasket 324. In some embodiments, each foot 344 projects from a comer of the gasket 324 (e.g., when two of the gasket walls 309 meet and form a right angle) or near a comer of the gasket 324. The feet 344 can be composed of a pliable or flexible material (e.g., a polymeric material such as silicone). In some embodiments, the feet 344 have a circular base having a diameter that is larger than a diameter of the holes 342. The feet 344 are configured to partially or completely deform in shape when being introduced into the holes 342 such that once inserted, the feet 344 are secured and not easily removed due to the difference in diameters of the circular base of the feet 344 and the holes 342. In some embodiments, the gasket 324 is permanently attached to the cover 336. In some embodiments, gasket 224 can be reversibly attached to cover 336.

Gasket 324 includes an L-shaped projection 326 extending from one of the gasket walls 309. In some embodiments, the L-shaped projection 326 extends from a comer of the gasket 324. In some embodiments, the L-shaped projection 326 is configured to be received by a recess 340 defined by the cover 336, In some embodiments, the recess 340 is an L- shaped recess. In some embodiments, the recess 340 has a shape that is complementary to the shape of the L-shaped projection 326.

The cover 336 includes a plurality of T-shaped ribs 346 and cross-shaped ribs 348 extending perpendicularly or protruding outwardly from the bottom surface 332 of cover 336. In some embodiments, the cover 336 includes eight T-shaped ribs 346. In some embodiments, the cover 336 includes at least about one to about eight T-shaped ribs 346 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 T-shaped ribs 346). The T-shaped ribs 346 can extend perpendicularly or protrude outwardly from a portion of the bottom surface 332 that is aligned with the length /_g of the gasket 324. The T-shaped ribs 346 can extend perpendicularly or protrude outwardly from a portion of the bottom surface 332 that is aligned with the width w_g of the gasket 324. In some embodiments, the cover 336 includes at least about one to about three cross-shaped ribs 348 (e.g., 1, 2 or 3 cross-shaped ribs 348). The cross-shaped ribs 348 can extend perpendicularly or protrude outwardly from a portion of the bottom surface 332 where four apertures 304 intersect.

FIG. 4 is a perspective view of the support device 400 including the substrate holder 401 and the substrate 410. The substrate holder 401 is shown as having the cover 436 detached from the base 434. The pair of hinges 416 can be breakable hinges. In some embodiments, the pair of hinges 416 are selectively, manually severable to permit a user to detach cover 436 from base 434. In some embodiments, the pair of hinges 416 can include a breakable portion. In some embodiments, the breakable portion can include breakable webbing. In some embodiments, the pair of hinges 416 is irreversibly breakable. For example, once broken, the cover 436 and base 434 cannot be attached via the pair of hinges 416. In some embodiments, the pair of hinges 416 may be reversibly breakable. For example, in some embodiments the pair of hinges 416 may include first portions 450 and second portions 452 that can be configured to reversibly engage with one another. In some embodiments, the first portions 450 of hinges 416 integrally extend from the cover 436 and the second portions 452 of hinge 416 integrally extend from the base 434. In some embodiments, the first and second portions 450, 452 can be directly engaged to each other via a fastener (e.g., a snap fit, force fit, or male and female connections). In some embodiments, no hinges 416 are present on the device.

In some embodiments, one or more sample regions 464 defined on the first surface 428 of substrate 410 can fall within the enclosed area defined by each one of the plurality of openings 438 of gasket 424. In some embodiments, substrate 410 may include samples (e.g., biological material samples) on a portion of its surface that align with one or more of the plurality of openings 438. In some embodiments, the samples (e.g., biological material samples) may be identified in the same manner as the corresponding aperture of the plurality of openings 438.

In some embodiments, the base 434 is configured to receive substrate 410. When the substrate holder 401 is in a closed position, the cover 436 is configured to be placed on top of the base 434 to secure the substrate 410 and the gasket 424 between the cover 436 and the base 434. In some embodiments, a user detaches the cover 436 from the base 434 before placing the substrate 410 in the base 434. Once detached, the user can insert the substrate 410 into the base 434 following the direction of the arrow 414. The user can place the cover 436 on top of the base 434 holding the substrate 410, e.g., in the direction of arrow 412, and snap the cover 436 onto the base 434 via the locking tabs 407 and 458 to secure the substrate 410.

In some examples, the second surface of the substrate 410 can rest on a portion of the base 434 of the substrate holder 401. For example, base 434 can include a lip 422 configured to retain the substrate 410 within the base 434. For example, substrate 410 can rest on lip 422 and be held in place. In some embodiments, lip 422 extends around the perimeter of the base 434. In some embodiments, the substrate can be secured to the base of the substrate holder. In an example, the substrate 410 can be placed in the base 434 in the direction of arrow 414. The second surface of substrate 410 can be placed such that it comes in contact with lip 222 and the first surface 428 of substrate 410 comes in contact with the plurality of ribs 418 and gasket 424 when the support device 400 is in a closed position. In some embodiments, substrate 410 can be loaded into base 434 without using a tool. The base 434 can include an opening 420 sufficiently sized to expose one or more portions of the substrate 410. In some embodiments, opening 420 is sized such that the majority of the second surface of substrate 410 is exposed and not covered. In some embodiments, opening 420 can enable the second surface of substrate 410 to come in contact with a surface of a heating device (e g., a plate 3110, as described in PCT/US2019/065100, the entire contents of which are incorporated herein by reference). In some embodiments, the first surface 428 of substrate 410 includes a sample region 464. In some embodiments, the base 434 includes opening 420 that exposes at least a portion of the second surface of the substrate 410 when the substrate 410 is placed in the base 434.

In some embodiments, moveable tabs 406 and 458 can have a C-shape structure. In some embodiments, moveable tabs 406 and 458 are constructed from a flexible material that enables them to be flexed away from the body of the substrate holder in order to engage the non-moveable tabs 408 and 462, respectively. In some embodiments, non-moveable tabs 408 and 462 are unable to be flexed. In some embodiments, non-moveable tabs 408 and 462 are rigid and do not flex when engaging the moveable tabs 406, 458.

In some embodiments, a first side wall 403 can measure about 3 inches. In some embodiments, a second side wall 405 can measure about 1 inch. In some embodiments, substrate 410 can measure about 75 millimeters (mm) by 25 mm. In some embodiments, substrate 410 can measure about 75 millimeters (mm) by 50 mm. In some embodiments, substrate 410 can measure about 48 millimeters (mm) by 28 mm. In some embodiments, substrate 410 can measure about 46 millimeters (mm) by 27 mm. In some embodiments, substrate 410 is a glass slide.

A support device may be substantially similar in construction and function in several aspects to the support device 100, 200, 300, 400 discussed above, but can include an alternative number, size, and shape of apertures defined by the cover and an alternative number, size, and shape of gasket openings instead of the plurality of apertures 104, 304 and the plurality of openings 138, 238, 338, 438. In some embodiments, the support device may have two apertures. In some embodiments, the support device may have two gasket openings. Such difference between the number, size, and shape of apertures and gasket openings can allow a user to use the substrate holder to support substrates having a wide variety of number, size, and shape of sample regions defined on the surface of the substrate.

FIG. 5 illustrates an example support device 500 in a closed position with two exemplary aperatures. The support device 500 includes a first aperture 554a and a second aperture 554b. The support device 500 includes two gasket openings that align with the first and second apertures 554a, 554b and enable portions of the substrate 510 to be exposed when the substrate holder 501 is in a closed position. In some embodiments, the support device 500 can include a cover 536 defining 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more apertures. In some embodiments, the support device 500 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more gasket openings. In some embodiments, the support device 500 can include a cover 536 defining a number of apertures ranging between 1 and 4, between 4 and 8, between 8 and 16, or between 16 and 20. In some embodiments, the support device 500 includes a number of gasket openings ranging between 1 and 4, between 4 and 8, between 8 and 16, or between 16 and 20.

In an aspect, the present disclosure includes a method of incubating a sample disposed on a sample region of any of the substrates disclosed herein. In some embodiments, the method includes mounting the substrate on any of the support devices disclosed herein. The method further includes positioning the substrate and support device on a heating apparatus (e.g., a laboratory heat plate). The method further includes activating the heating apparatus to transfer heat to the sample (e.g., via the second surface of the substrate that is exposed via the opening of the substrate holder). In some embodiments, when the substrate holder is coupled to the support device, at least 60% of a sample region is overlaid by the support device. In some embodiments, at least 50%, 40%, 30%, 20%, 10% or less of a sample region of a substrate is overlaid by the support device.

While embodiments shown in FIGS. 1-4 show non-moveable tabs (e.g., the nonmoveable tabs 108, 160, 208, 260, 408, 462) on the base (e.g., the base 134, 234, 334, 434) and the moveable tabs (e.g., the moveable tabs 106, 162, 206, 262, 406, 460) on the cover (e.g., the cover 136, 236, 436), in further embodiments, the non-moveable tabs are on the cover and the moveable tabs are on the base.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims.

Disclosed are systems, apparatuses (e.g., devices), and methods that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods. These and other systems, apparatuses, and methods are disclosed herein, and it is understood that combinations, subsets, interactions, groups, etc. of these systems, apparatuses, and methods are disclosed. That is, while specific reference to each various individual and collective combinations and permutations of these systems, apparatuses and methods may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular system, apparatus, or a particular method is disclosed and discussed and a number of systems, apparatuses, or methods are discussed, each and every combination and permutation of the systems, apparatuses, and the methods are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed.

Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A support device comprising: a substrate holder comprising: a cover comprising opposing first side walls, the cover configured to receive a gasket; a base comprising opposing first side walls, the base configured to receive a sample substrate; and a first locking tab and a second locking tab, the first and second locking tabs comprising moveable tabs coupled to the first side walls of one of the cover and the base and non-moveable tabs coupled to the first side walls of the other of the cover and the base, the first locking tab having a length greater than a length of the second locking tab, wherein the first locking tab extends along a length of the gasket, and wherein the moveable tabs are configured to engage with the non-moveable tabs to releasably secure the cover to the base.

2. The support device of claim 1, wherein a height of the first locking tab is about equal to a height of the gasket.

3. The support device of claim 1, wherein the length of the first locking tab is about equal to the length of the gasket.

4. The support device of claim 1, wherein the sample substrate rests on top of the base of the substrate holder.

5. The support device of claim 1, wherein the cover comprises a plurality of ribs extending from a surface of the cover, and wherein the gasket is positionally aligned on the surface of the cover by the plurality of ribs.

6. The support device of claim 1, wherein the gasket is positioned so that when the sample substrate is secured by the substrate holder, and the substrate holder is in a closed position, a vapor-tight seal is formed between the gasket and the sample substrate.

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7. The support device of claim 1, wherein the cover comprises at least two apertures.

8. The support device of claim 7, wherein the gasket comprises at least two openings, wherein the at least two openings are positioned so that when the sample substrate is secured by the substrate holder, and the substrate holder is in a closed position, the at least two apertures are aligned with the at least two openings.

9. The support device of claim 8, wherein the gasket is configured to prevent fluid transport between the at least two openings when the cover is in the closed position.

10. The support device of claim 1, wherein the base comprises a lip extending around a perimeter of the base, the lip configured to retain the sample substrate within the base.

11. The support device of claim 1, wherein the cover comprises a plurality of ribs extending from a surface of the cover, and wherein the sample substrate engages with at least one of the plurality of ribs when the substrate holder is in a closed position.

12. The support device of claim 1, wherein the base comprises an opening exposing at least a portion of the sample substrate when the sample substrate is placed in the base.

13. The support device of claim 1, wherein each of the first side walls of the cover is generally orthogonal to a second side wall of the cover.

14. The support device of claim 1, wherein when the substrate holder is in a closed position, the cover is configured to fold onto the base.

15. The support device of claim 1, wherein the length of the first locking tab is at least 90% of the length of the gasket.

16. A support device comprising: a substrate holder comprising: a cover comprising opposing first side walls and opposing second side walls, the cover configured to receive a gasket;

34 a base comprising opposing first side walls and opposing second side walls, the base configured to receive a sample substrate, each of the first side walls of the base and the cover being longer than each of the second side walls of the base and the cover; and a locking tab comprising a moveable tab and a non-moveable tab, one of the moveable tab and the non-moveable tab coupled to one of the first side walls of the cover, and another of the moveable tab and the non-moveable tab coupled to one of the first side walls of the base, the locking tab having a length about equal to a length of the gasket, wherein the locking tab extends along the length of the gasket.

17. The support device of claim 16, wherein the sample substrate rests on top of the base of the substrate holder.

18. The support device of claim 16, wherein the cover comprises a plurality of ribs extending from a surface of the cover, and the gasket is positionally aligned on the surface of the cover by the plurality of ribs.

19. The support device of claim 16, wherein the gasket is positioned so that when the sample substrate is secured by the substrate holder, and the substrate holder is in a closed position, a vapor-tight seal is formed between the gasket and the sample substrate.

20. The support device of claim 16, wherein the cover comprises one aperture.

21. The support device of claim 16, wherein the cover comprises at least two apertures.

22. The support device of claim 21 , wherein the gasket comprises at least two openings, wherein the at least two openings is positioned so that when the sample substrate is secured by the substrate holder and the substrate holder is in a closed position, the at least two apertures is aligned with the at least two openings.

23. The support device of claim 22, wherein the gasket is configured to prevent fluid transport between the at least two openings when the substrate holder is in the closed position.

24. The support device of claim 16, wherein the sample substrate comprises a glass slide.

25. The support device of claim 16, wherein the base comprises a lip extending around a perimeter of the base, the lip configured to retain the sample substrate within the base.

26. The support device of claim 16, wherein the cover comprises a plurality of ribs extending from a surface of the cover, and the sample substrate engages wi th at least one of the plurality of ribs when the substrate holder is in a closed position.

27. The support device of claim 16, wherein the base comprises an opening exposing at least a portion of the sample substrate when the sample substrate is placed in the base.

28. The support device of claim 16, wherein each of the first side walls of the cover is generally orthogonal to the second side walls of the cover.

29. The support device of claim 16, further comprising at least one breakable hinge extending from one of the second side walls of the cover to one of the second side walls of the base such that, when the substrate holder is in a closed position, the cover is configured to fold onto the base to secure the sample substrate and the gasket between the cover and the base.

30. The support device of claim 29, wherein the cover and the base are pivotably connected by at least one hinge extending from one of the second side walls of the cover to one of the second side walls of the base such that when the substrate holder is in the closed position, the cover configured to fold over the base to secure the sample substrate and the gasket between the cover and the base.

31. The support device of claim 29, wherein the cover and the base are pivotably connected by the at least one breakable hinge.

32. A method of incubating a sample disposed on a sample region of a substrate, the method comprising: mounting the substrate on a base of a substrate holder of a support device, the substrate comprising the sample; moving a cover of the substrate holder toward the base of the substrate holder such that first and second moveable tabs on one of the base and the cover engage with first and second non-moveable tabs on the other of the base and the cover, the first moveable tab having a length greater than a length of the second moveable tab and extending along a length of a gasket received by the cover; positioning the substrate and the support device on a heating apparatus; and activating the heating apparatus to transfer heat to the sample.

33. The method of claim 32, wherein the substrate comprises a glass slide.

34. The method of claim 32, wherein, when the substrate holder is coupled to the support device, at least 60% of the sample region is overlaid by the support device.

35. The method of claim 32, wherein a height of the first moveable tab is about equal to a height of the gasket.

36. The method of claim 32, wherein a length of the first moveable tab is about equal to a length of the gasket.

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