Imaging station with system for aligning and registering a sample device

Abstract

A system for precise and repeatable positioning of a cassette, e.g., for fluorescence imaging of a sample disposed on a substrate, includes a moveable stage configured for motion in an X-direction and Y-direction and a chuck disposed on the moveable stage. The chuck is configured to receive a substrate secured within the cassette, and the chuck has a first side, a second side opposite the first side, and a top surface. The chuck includes at least one magnetic component disposed in or on the chuck. The at least one magnetic component in the chuck is configured to magnetically couple to at least one magnetic component in the cassette when the cassette is positioned on the chuck.

Description

GEI-03525IMAGING STATION WITH SYSTEM FOR ALIGNING AND REGISTERING A SAMPLE DEVICECROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional App. No. 63 / 740,019, filed December 30, 2024. The entire contents of which is hereby incorporated by reference.FIELD

[0002] The present disclosure relates to an imaging station with a system for holding a biological sample positioned within a sample device (e.g., a cassette). Particularly, the present disclosed subject matter is directed to an imaging station configured to align and register a sample device.BACKGROUND

[0003] Many biomedical applications rely on imaging many biological samples rapidly to produce highly accurate data. For instance, in both research and clinical applications, large volumes of biological samples enclosed in cassettes may be loaded into imaging stations in succession. However, issues can occur in these cases where the cassette may be misregistered or misaligned with various imaging station instruments, resulting in blurry images or inaccurate data being collected. Conventional systems include securing means which take up excess space or require unnecessary strain and time to fix a sample enclosed in a cassette into an imaging station.

[0004] Thus, there exists a need in the art for an improved imaging station system with a mechanism for aligning, registering, and securely holding sample devices enclosed in cassette casings and aligning them with imaging station instruments.SUMMARY

[0005] The purpose and advantages of the disclosed subject matter will be set forth in and apparent from the description that follows, as well as will be learned by practice of the disclosed subject matter. Additional advantages of the disclosed subject matter will be realized and attained by the methods and systems particularly pointed out in the written description and claims hereof, as well as from the appended drawings.

[0006] To achieve these and other advantages and in accordance with the purpose of the disclosed subject matter, as embodied and broadly described, the disclosed subject matter includes a system comprising: a moveable stage configured for motion in an X-direction and Y-direction; a pedestal disposed on the moveable stage, the pedestal configured to receive aFH12653613.3substrate secured within a cassette, the pedestal having a first side, a second side opposite the first side, and a top surface; and at least one magnetic component disposed relative to (e.g., on, in, partially within, adjacent to) the pedestal. In some embodiments, the magnetic component can be entirely incorporated into the pedestal, and / or have portion(s) located outside the pedestal.

[0007] In some embodiments, the system comprises a frame configured to support an optical train, wherein the moveable stage is coupled to the frame.

[0008] In some embodiments, the first side is a first long side and the second side is a second long side, the pedestal further comprising a first short side and a second short side opposite the second short side.

[0009] In some embodiments, the pedestal comprises a first side recess on the first side and a second side recess on the second side.

[0010] In some embodiments, the at least one magnetic component comprises a first magnetic component and a second magnetic component.

[0011] In some embodiments, the first magnetic component is disposed within the first side recess and the second magnetic component is disposed within the second side recess.

[0012] In some embodiments, the at least one magnetic component is arranged parallel to the first side or the second side.

[0013] In some embodiments, the at least one magnetic component in or on the pedestal is arranged to magnetically couple with at least one magnetic component in the cassette when the cassette is positioned on the pedestal.

[0014] In some embodiments, the pedestal comprises at least one seating member extending from the top surface and configured to engage the substrate, wherein the seating member has a substantially planar seating surface.

[0015] In some embodiments, wherein the at least one seating member is continuous about a perimeter of the top surface of the pedestal.

[0016] In some embodiments, the at least one seating member is discontinuous about a perimeter of the top surface of the pedestal.

[0017] In some embodiments, the at least one seating member comprises a three-point support.-2-FH12653613.3

[0018] In some embodiments, the three-point support comprises a first seating member and a second seating member on a third side of the pedestal and a third seating member on a fourth side of the pedestal.

[0019] In some embodiments, the pedestal comprises a chamfer between the first side, the second side, and the top surface, wherein the chamfer is configured to engage with a complementary angled surface on a bottom surface of the cassette.

[0020] In some embodiments, the pedestal has a first width from the first side to the second side, and the top surface has a second width that is less than the first width.

[0021] In some embodiments, the system further comprises a cassette presence sensor configured to detect the presence and / or absence of a cassette on the pedestal.

[0022] In some embodiments, the at least one magnetic component comprises a ferromagnetic material.

[0023] In some embodiments, the ferromagnetic material comprises iron, alloy steel, stainless steel, nickel, cobalt, gadolinium, neodymium, ferromagnetic ceramic, or a combination thereof.

[0024] In some embodiments, the system further comprises the cassette and the substrate, wherein the substrate includes a sample region configured to receive at least one sample, wherein the cassette comprises a top portion and a bottom portion and the substrate is secured therebetween, and wherein the cassette defines an open well around the sample region.

[0025] In various embodiments, a method is provided. The method comprises the following steps. A cassette is positioned on the pedestal of the system to thereby magnetically couple the at least one magnetic component in or on the pedestal to at least one magnetic component in the cassette, wherein the cassette comprises a top portion, a bottom portion, and a substrate secured therebetween, wherein the substrate includes a sample region configured to receive at least one sample, and wherein the cassette defines an open well around the sample region. The cassette is removed from the pedestal.

[0026] In some embodiments, the method further comprises imaging the at least one sample in the open well after positioning the cassette on the pedestal.

[0027] In some embodiments, the method further comprises a robotic arm configured to position the cassette on the pedestal and / or remove the cassette from the pedestal.-3- FH12653613.3

[0028] In some embodiments, removing the cassette from the pedestal comprises moving the cassette vertically up and away from the pedestal to thereby magnetically decouple the at least one magnetic component in the pedestal from the at least one magnetic component in the pedestal.

[0029] The disclosed subject matter also includes a system comprising a frame, a moveable stage coupled to the frame and configured for motion in an X-direction and Y-direction, and a chuck disposed on the moveable stage. The chuck is configured to receive a substrate secured within a cassette casing. The chuck has a first end, a second end, opposing sides, a top surface, and at least one fluidic channel therein configured to apply a vacuum pressure between the sample substrate and the top surface of the chuck. The system further includes at least one casing registration feature disposed on the moveable stage and configured to align the cassette casing. The at least one casing registration feature includes a XY constraint and a X constraint. An alignment mechanism is included in the system. The moveable stage is displaced from a first position where the cassette casing is spaced from the casing registration feature to a second position where the cassette casing is engaged with the alignment mechanism and the casing registration feature when the moveable stage translates in at least one of the X-direction and Y-direction.

[0030] In some embodiments, the XY constraint comprises a V-groove.

[0031] In some embodiments, the X constraint comprises a substantially flat portion.

[0032] In some embodiments, the X constraint is offset from the XY constraint by a distance.

[0033] In some embodiments, the X constraint and the XY constraint are positioned along an axis parallel to a Y axis of the moveable stage.

[0034] In some embodiments, the alignment mechanism is fixed with respect to the frame.

[0035] In some embodiments, the alignment mechanism is not on the stage.

[0036] In some embodiments, a portion of the alignment mechanism extends in the X-direction and / or Y-direction beyond the moveable stage.

[0037] In some embodiments, the alignment mechanism comprises a ball plunger.

[0038] In some embodiments, the ball plunger is aligned perpendicular to the at least one cassette registration feature.-4-FH12653613.3

[0039] In some embodiments, displacing the movable stage from the first position to the second position engages the casing with the alignment mechanism at a midpoint of a side of the casing.

[0040] In some embodiments, the alignment mechanism is disposed on the moveable stage.

[0041] In some embodiments, the alignment mechanism includes a rotatable cam portion which displaces the alignment mechanism in the X-direction.

[0042] In some embodiments, the rotatable cam portion is coupled to an actuator.

[0043] In some embodiments, the actuator comprises an electric motor.

[0044] In some embodiments, the actuator comprises a pneumatic actuator.

[0045] In some embodiments, the first pressure is applied to the chuck during movement of the moveable stage from the first position to the second position.

[0046] In some embodiments, the chuck includes a heat transfer device.

[0047] In some embodiments, the at least one casing registration feature is configured to receive at least one kinematic alignment feature disposed on a surface of the cassette casing.

[0048] In some embodiments, each of the at least one kinematic alignment feature of the cassette casing comprises a rigid spherical portion.

[0049] In some embodiments, displacing the movable stage from the first position to the second position provides an X-force on the cassette casing to thereby register the cassette casing with at least one casing registration feature.

[0050] In some embodiments, the opposing sides of the chuck include recesses configured to receive tines of a forklift.

[0051] In some embodiments, the movable stage displaces the casing to align the sample substrate with an imaging device along the Z-axis.

[0052] In some embodiments, the imaging device is at least partially disposed within the casing.

[0053] In various embodiments, a method is provided. The method comprises the following steps. The system described above is provided and the substrate is positioned on the chuck. The substrate is secured within the cassette casing, and the cassette casing comprises at least one kinematic alignment feature disposed on its surface. The moveable-5- FH12653613.3stage and / or the alignment mechanism is actuated to thereby align the at least one kinematic alignment feature with the at least one casing registration feature.

[0054] In some embodiments, the method further comprises applying a first pressure to the chuck when the cassette casing is disposed thereon and applying a second pressure when the moveable stage is in the second position.

[0055] In some embodiments, the first pressure is less than the second pressure, and the second pressure is configured to maintain the substrate in a substantially parallel orientation with an XY plane defined by an upper surface of the chuck.

[0056] In various embodiments, a method is provided. The method comprises the following steps. A system comprising a frame is provided. A moveable stage is coupled to the frame and configured for motion in an X-direction and Y-direction. A chuck is disposed on the moveable stage and is configured to receive a substrate secured within a cassette casing. The chuck may have a first end, a second end, opposing sides, a top surface, and at least one fluidic channel therein configured to apply a vacuum pressure between the sample substrate and the top surface of the chuck. At least one casing registration feature may be disposed on the moveable stage and configured to align with the cassette casing. The at least one casing registration feature may include a XY constraint and a X constraint. An alignment mechanism is be positioned on the frame, wherein the alignment mechanism is fixed with respect to the frame and extends in the X-direction and / or Y-direction beyond the moveable stage. The substrate may be positioned on the chuck and secured within the cassette casing. The cassette casing may comprise at least one kinematic alignment feature disposed on a surface of the cassette casing. The moveable stage may be actuated from a first position where the cassette casing is spaced from the casing registration feature to a second position where the cassette casing is engaged with the alignment mechanism and the at least one kinematic alignment feature is engaged with the at least one casing registration feature.

[0057] In some embodiments, the alignment mechanism comprises a ball plunger aligned perpendicular to the at least one cassette registration feature.

[0058] In some embodiments, the method further comprises applying a first pressure to the chuck when the cassette casing is disposed thereon and applying a second pressure when the moveable stage is in the second position.-6- FH12653613.3

[0059] In some embodiments, the first pressure is less than the second pressure, and the second pressure is configured to maintain the substrate in a substantially parallel orientation with an XY plane defined by an upper surface of the chuck.

[0060] It is to be understood that both the foregoing general description and the following detailed description are exemplary and are intended to provide further explanation of the disclosed subject matter claimed.

[0061] The accompanying drawings, which are incorporated in and constitute part of this specification, are included to illustrate and provide a further understanding of the method and system of the disclosed subject matter. Together with the description, the drawings serve to explain the principles of the disclosed subject matter.BRIEF DESCRIPTION OF THE DRAWINGS

[0062] A detailed description of various aspects, features, and embodiments of the subject matter described herein is provided with reference to the accompanying drawings, which are briefly described below. The drawings are illustrative and are not necessarily drawn to scale, with some components and features being exaggerated for clarity. The drawings illustrate various aspects and features of the present disclosure and may illustrate one or more embodiment(s) or example(s) of the present disclosure in whole or in part.

[0063] FIG. 1A is a view of an exemplary imaging station assembly configuration, with the cassette casing and a substrate held within, in accordance with the present disclosure.

[0064] FIG. IB is a view of the exemplary imaging station assembly, without a cassette casing and substrate held within, in accordance with the present disclosure.

[0065] FIG. 1C is a top view of the exemplary imaging station assembly without the cassette casing for holding a substrate, in accordance with the present disclosure.

[0066] FIG. ID is a side view of the exemplary imaging station assembly with the cassette casing for holding a substrate, in accordance with the present disclosure.

[0067] FIG. 2 is a top view of the casing registration feature, cassette casing, and kinematic alignment features in accordance with the present disclosure.

[0068] FIG. 3 A is a side view of the exemplary imaging station assembly with vacuum chuck and heat transfer device in accordance with the present disclosure.

[0069] FIG. 3B is a perspective exploded view of the vacuum chuck and heat transfer device in accordance with the present disclosure.-7-FH12653613.3

[0070] FIG. 4A is a view of the exemplary imaging station assembly holding a cassette casing and sample and a first exemplary off-stage alignment mechanism, in accordance with the present disclosure.

[0071] FIG. 4B is a view of an exemplary embodiment of the imaging station with a first exemplary off-stage alignment mechanism in accordance with the present disclosure.

[0072] FIG. 4C is a partial view of an exemplary embodiment of the imaging station with a first exemplary off-stage alignment mechanism in accordance with the present disclosure.

[0073] FIG. 4D is a front (Y-axis) view of an exemplary embodiment of the imaging station in proximity to the first exemplary off-stage alignment mechanism in accordance with the present disclosure.

[0074] FIG. 4E is a partial view of the imaging station assembly and frame with a first exemplary off-stage alignment mechanism and off-stage components in accordance with the present disclosure.

[0075] FIG. 5A is a view of an exemplary embodiment of the imaging station with a second exemplary on-stage alignment mechanism in accordance with the present disclosure.

[0076] FIG. 5B is a side view of an exemplary imaging station with a second exemplary on-stage alignment mechanism in accordance with the present disclosure.

[0077] FIG. 5C is a partial top view of an exemplary imaging station with a second exemplary on-stage alignment mechanism in accordance with the present disclosure.

[0078] FIG. 6 A is a view of the cassette casing and transfer mechanism loading the cassette casing into the imaging station in accordance with the present disclosure.

[0079] FIG. 6B is a view of the cassette casing and transfer mechanism loading the cassette casing into the imaging station in accordance with the present disclosure.

[0080] FIG. 7A is a partial view of the imaging station assembly and loading station with a first exemplary off-stage alignment mechanism in accordance with the present disclosure.

[0081] FIG. 7B is a side view of the imaging station assembly and imaging device in accordance with the present disclosure.

[0082] FIG. 7C is a side view of the cassette casing being removed from the imaging station assembly and imaging device in accordance with the present disclosure.-8-FH12653613.3

[0083] FIG. 8 A is a top view of the printed circuit board assembly in accordance with the present disclosure.

[0084] FIG. 8B is a top view of the cable routing of the exemplary imaging station assembly in accordance with the present disclosure.

[0085] FIG. 9 is a top view of the first exemplary imaging station assembly, with an off-stage alignment mechanism, enclosed within a larger processing system in accordance with the present disclosure.

[0086] FIG. 10 is a top view of the second exemplary imaging station assembly, with an on-stage alignment mechanism, enclosed within a larger processing system in accordance with the present disclosure.

[0087] FIG. 11 depicts various cassette presence sensor options suitable for use with the imaging station assembly in accordance with the present disclosure.

[0088] FIG. 12 depicts a pedestal having a three-point contact mechanism for aligning a sample device (e.g., a cassette assembly) in accordance with the present disclosure.

[0089] FIG. 13A illustrates an imaging station having a moveable stage with a pedestal having magnetic components for magnetically coupling magnetic components in a sample device (e.g., a cassette assembly) in accordance with the present disclosure. FIGS.13B-13C illustrate views of the pedestal of FIG. 13A.

[0090] FIGS. 14A-14C depicts various cross-sectional views of a system including a sample device (e.g., cassette assembly) having a base with magnetic components positioned on a pedestal having corresponding magnetic components in accordance with the present disclosure.DETAILED DESCRIPTION

[0091] Definitions

[0092] To facilitate the understanding of this disclosure, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the disclosure. Terms such as “a”, “an,” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the disclosure, but their usage does not limit the disclosure, except as outlined in the claims.-9-FH12653613.3

[0093] Where values are described as ranges, it will be understood that such disclosure 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.

[0094] The term “about,” as used herein, refers to ±10% of a recited value.

[0095] As used herein, any values provided in a range of values include both the upper and lower bounds, and any values contained within the upper and lower bounds.

[0096] The term “biological particle,” as used herein, generally refers to a discrete biological system derived from a biological sample. The biological particle may be a virus. The biological particle may be a cell or derivative of a cell. The biological particle may be an organelle from a cell. Examples of an organelle from a cell include, without limitation, a nucleus, endoplasmic reticulum, a mitochondrion, a ribosome, a Golgi apparatus, an endoplasmic reticulum, a chloroplast, an endocytic vesicle, an exocytic vesicle, a vacuole, and a lysosome. The biological particle may be a rare cell from a population of cells. The biological particle may be any type of cell, including without limitation prokaryotic cells, eukaryotic cells, bacterial, fungal, plant, mammalian, or other animal cell type, mycoplasmas, normal tissue cells, tumor cells, or any other cell type, whether derived from single cell or multicellular organisms. The biological particle may be a constituent of a cell. The biological particle may be or may include DNA, RNA, organelles, proteins, or any combination thereof. The biological particle may be or may include a matrix (e.g., a gel or polymer matrix) including a cell or one or more constituents from a cell (e.g., cell bead), such as DNA, RNA, organelles, proteins, or any combination thereof, from the cell. The biological particle may be obtained from a tissue of a subject. The biological particle may be a hardened cell. Such hardened cell may or may not include a cell wall or cell membrane. The biological particle may include one or more constituents of a cell but may not include other constituents of the cell. An example of such constituents is a nucleus or an organelle. A cell may be a live cell. The live cell may be capable of being cultured, for example, being cultured when enclosed in a gel or polymer matrix or cultured when including a gel or polymer matrix.

[0097] The term “fluidically connected”, as used herein, refers to a direct connection between at least two device elements, e.g., a channel, reservoir, etc., that allows for fluid to move between such device elements without passing through an intervening element.-10-FH12653613.3

[0098] The term “genome,” as used herein, generally refers to genomic information from a subject, which may be, for example, at least a portion or an entirety of a subject’s hereditary information. A genome can be encoded either in DNA or in RNA. A genome can include coding regions that code for proteins as well as non-coding regions. A genome can include the sequence of all chromosomes together in an organism.

[0099] For example, the human genome has a total of 46 chromosomes. The sequence of all of these together may constitute a human genome.

[0100] The term “in fluid communication with”, as used herein, refers to a connection between at least two device elements, e.g., a channel, reservoir, etc., that allows for fluid to move between such device elements with or without passing through one or more intervening device elements.

[0101] The term “macromolecular constituent,” as used herein, generally refers to a macromolecule contained within or from a biological particle. The macromolecular constituent may include a nucleic acid. In some cases, the biological particle may be a macromolecule. The macromolecular constituent may include DNA or a DNA molecule. The macromolecular constituent may include RNA or an RNA molecule. The RNA may be coding or non-coding. The RNA may be messenger RNA (mRNA), ribosomal RNA (rRNA) or transfer RNA (tRNA), for example. The RNA may be a transcript. The RNA molecule may be (i) a clustered regularly interspaced short palindromic (CRISPR) RNA molecule (crRNA) or (ii) a single guide RNA (sgRNA) molecule. The RNA may be small RNA that are less than 200 nucleic acid bases in length, or large RNA that are greater than 200 nucleic acid bases in length. Small RNAs may include 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNAs), Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA) and small rDNA-derived RNA (srRNA). The RNA may be double-stranded RNA or singlestranded RNA. The RNA may be circular RNA. The macromolecular constituent may include a protein. The macromolecular constituent may include a peptide. The macromolecular constituent may include a polypeptide or a protein. The polypeptide or protein may be an extracellular or an intracellular polypeptide or protein. The macromolecular constituent may also include a metabolite. These and other suitable macromolecular constituents (also referred to as analytes) will be appreciated by those skilled in the art (see U.S. Pat. Nos. 10,011,872-11-FH12653613.3and 10,323,278, and PCT Publication No. WO 2019 / 157529, each of which is incorporated herein by reference in its entirety).

[0102] The term “particulate component of a cell” refers to a discrete biological system derived from a cell or fragment thereof and having at least one dimension of 0.01 pm (e.g., at least 0.01 pm, at least 0.1 pm, at least 1 pm, at least 10 pm, or at least 100 pm). A particulate component of a cell may be, for example, an organelle, such as a nucleus, an exosome, a liposome, an endoplasmic reticulum (e.g., rough or smooth), a ribosome, a Golgi apparatus, a chloroplast, an endocytic vesicle, an exocytic vesicle, a vacuole, a lysosome, or a mitochondrion.

[0103] The terms “sample,” “tissue sample,” and “biological tissue sample” as used herein, refers to material from a subject, such as a biopsy, core biopsy, tissue section, needle aspirate, or fine needle aspirate or skin sample. The biological tissue sample may be derived from another sample. The biological sample may be a nucleic acid sample or protein sample. The sample may be a liquid sample, such as a blood sample, urine sample, or saliva sample. The sample may be a skin sample. The sample may be a cheek swap. The sample may be a plasma or serum sample. The sample may include a biological particle, e.g., a cell or virus, or a population thereof, or it may alternatively be free of biological particles. A cell-free sample may include polynucleotides. Polynucleotides may be isolated from a bodily sample that may be selected from the group consisting of blood, plasma, serum, urine, saliva, mucosal excretions, sputum, stool, and tears.

[0104] The term “sequencing,” as used herein, generally refers to methods and technologies for determining the sequence of nucleotide bases in one or more polynucleotides. The polynucleotides can be, for example, nucleic acid molecules such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including variants or derivatives thereof (e.g., single stranded DNA). Sequencing can be performed by various systems currently available, such as, without limitation, a sequencing system by ILLUMINA®, Pacific Biosciences (PACBIO®), Oxford NANOPORE®, or Life Technologies (ION TORRENT®). Alternatively, or in addition, sequencing may be performed using nucleic acid amplification, polymerase chain reaction (PCR) (e.g., digital PCR, quantitative PCR, or real time PCR), or isothermal amplification. Such systems may provide a plurality of raw genetic data corresponding to the genetic information of a subject (e.g., human), as generated by the systems from a sample provided by the subject. In some examples, such systems provide -12-FH12653613.3sequencing reads (also “reads” herein). A read may include a string of nucleic acid bases corresponding to a sequence of a nucleic acid molecule that has been sequenced. In some situations, systems and methods provided herein may be used with proteomic information.

[0105] The term “subject,” as used herein, generally refers to an animal, such as a mammal (e.g., human) or avian (e.g., bird), or other organism, such as a plant. The subject can be a vertebrate, a mammal, a mouse, a primate, a simian or a human. Animals may include, but are not limited to, farm animals, sport animals, and pets. A subject can be a healthy or asymptomatic individual, an individual that has or is suspected of having a disease (e.g., cancer) or a pre-disposition to the disease, or an individual that is in need of therapy or suspected of needing therapy. A subject can be a patient.

[0106] The term “inlet” and “port” as used herein, generally refers to an aperture, orifice or channel extending through at least a portion of a device layer.

[0107] Reference will now be made in detail to exemplary embodiments of the disclosed subject matter, an example of which is illustrated in the accompanying drawings. The method and corresponding steps of the disclosed subject matter will be described in conjunction with the detailed description of the system.

[0108] The methods, assemblies, and systems presented herein relate to an imaging station configured to receive, align, register, and / or secure a sample device (e.g., a cassette forming an open well) having a sample (e.g., a biological sample) positioned therein. In some embodiments, the sample device is secured within a casing having one or more alignment and / or registration features on an exterior surface thereof. The one or more alignment and / or registration features on the casing are configured to contact complementary alignment and / or registration features on the imaging station to provide for repeatable and reliable alignments and registration of the sample device in the imaging station, thereby allowing for the sample device to be moved to and from the imaging station during a plurality of probing-imaging cycles (e.g., 15 to 35 cycles) of an in situ analysis run.

[0109] Assembly 100

[0110] FIGS. 1A - FIG. ID are various views of an imaging station 100 holding (FIG. 1A), and without for clarity (FIG. IB), a cassette casing 102. FIG. 1A and FIG. ID depict an imaging station 100 assembly holding a biological sample disposed on a substrate that is enclosed in a cassette casing 102. As shown in FIG. 1A and FIG. ID, an imaging station assembly 100 is configured to hold a cassette casing 102 with a sample device 114.-13-FH12653613.3The cassette casing 102 includes a casing lid 102f and a casing base 103f each having an upper surface and a bottom surface defining a thickness therebetween and opposing sides. The casing 102 includes a length- wise side 102a formed by the casing lid 102f and a lengthwise side 103a formed by the casing base 103f. Furthermore, two width- wise sides 102b, 103b are formed by the casing lid 102f and base 103f, respectively. The cassette casing assembly can be re-usable. The casing lid 102f and casing base 103f can be substantially rectangular in shape and formed of a rigid material (e.g., metal, plastic), with the lid being removable from the base. In some embodiments, the lid 102f is hingedly attached to the base 103f so that the lid can pivot or rotate into an open position to receive the substrate and supporting sample device 114 therein. The cassette casing 102 and sample are disposed within an imaging station assembly 100. The casing base 103 may include a recess, defining an interior surface for receiving a sample device 114 which may hold a biological sample / substrate to be processed in the imaging station. As will be discussed in more detail below, some embodiments of the system may forgo a cassette casing 102 entirely and directly align and register the sample device 114 (e.g., a cassette) to the imaging station 100.

[0111] FIG. IB and FIG. 1C are a perspective and top view of the imaging station 100 without a cassette casing 102 (omitted for clarity). An exemplary imaging station 100 includes a moveable stage 101 wherein a plurality of components may be situated on top. The moveable stage 101 may comprise an upper and lower surface, defining a thickness therebetween and form a length-wise sidewall 101a and a width-wise side wall 101b. The moveable stage may be substantially rectangular in shape or may comprise any suitable shape based on the desired arrangement of components and their dimensions. A plurality of apertures 116a-116d may be disposed along the surface of the moveable stage. These may be utilized to pass through a fastener or any suitable type (e.g. bolts, rivets, screws, etc.) in order to secure the moveable stage 101 to other components.

[0112] Referring to FIG. IB and FIG. 1C, a casing holder 109 may be disposed on the moveable stage 101 and may be configured to receive the cassette casing 102. The casing holder may include a chuck 109 (which may also be referred to as a “pedestal”). The chuck 109 may have a first end, a second end, opposing sides, and a top surface. The chuck 109 may possess a “bench”-like shape, with a first leg 109a and second leg 109b on top of which a rectangular top surface 109c rests upon. The top surface 109c can include a seating member 111 (e.g., which may take the form of one or more planar extrusions), defining a region -14-FH12653613.3configured to receive a sample / substrate (which is secured within a cassette casing). That is, the bottom surface of the substrate (e.g. glass slide containing the sample) contacts the upper surface of the seating member 111 of the chuck while the cassette casing 102, and sample device 114 contained therein, can be spaced from the seating member 111 (as described in further detail herein in connection with Fig. 3B). In some embodiments, the seating member 111 includes a single surface for engaging the substrate and / or the sample device (e.g., a cassette). In some embodiments, the seating member 111 includes a plurality of surfaces for engaging the substrate and / or the sample device (e.g., a cassette).

[0113] The seating memberl 11 can be received within a corresponding aperture in the cassette casing base 103 to contact the substrate. In some embodiments, the chuck 109 provides a vacuum pressure for suctioning the lower surface of the substrate to the surface of the chuck through at least one fluidic channel Illa which opens onto the upper surface of the chuck. In the exemplary embodiment shown, the fluidic channel Illa circumscribes the seating member 111 to provide a vacuum force around the entire perimeter of the substrate.A vacuum inlet port 113 may be located at one exterior side of the chuck for attachment to a vacuum source (not shown), which may be for example but not limited to a vacuum pump. Heat transfer devices 130, which are described in further detail below, may be included within the chuck to maintain a sample temperature of approximately ~23°C + / - 2°C.

[0114] In some embodiments, the chuck 109 and heat transfer device 130 may be configured to maintain a temperature suitable based on whichever biological sample is loaded into the cassette casing 102 and onto the chuck 109.

[0115] The chuck 109 may be secured to the surface of the base 101 through a variety of suitable methods. In some embodiments, the chuck may possess a plurality of apertures 112 at each comer, which extend into the supporting legs, defining a space for a suitable fastener to extend through (e.g. screw, nut, bolt, rivet). However, the chuck securing mechanism is not limited to the described mechanism. For example, and without limitation, the chuck may also be secured through any suitable joining mechanism, such as welding, adhesive bonding, or magnetic coupling to the base 101.

[0116] At least one casing registration feature 104 may also be disposed on the surface of the base 101, in a position adjacent to a side of the cassette casing 102. The casing registration 104 feature may be an elongated body, with a height suitable such that the top surface is aligned and coplanar with the top surface of the cassette casing 102. The casing -15-FH12653613.3registration feature may be substantially rectangular such that the side facing the cassette casing 104a may match the contour of the side of the cassette casing 102. In the exemplary embodiment shown, the registration feature 104 extends in the Y-direction a distance beyond the chuck 109 (so that the chuck 109 and registration feature 104 are offset or not aligned with respect to the X-direction).

[0117] In some embodiments, the casing registration feature 104 may possess a plurality of apertures 115a, 115b, defining a space for a suitable fastener (e.g. bolt, screw, rivet) to extend through, in order to secure the feature to the base 101. The apertures 115a, 115b may be spaced apart from one another in the vertical direction. This allows for the registration feature 104 to be repositioned with respect to the moveable stage 101, as desired. In other embodiments, the casing registration feature 104 may be joined to the surface of the base 101 by welding, adhesive bonding, or magnetic coupling.

[0118] In some embodiments, the casing registration feature may include an XY constraint 105 comprising a V-groove on the surface of the exterior side wall 104a of the casing registration feature. In some embodiments, the casing registration feature may include a X constraint 106 comprising a substantially flat surface on the surface of the exterior side wall of the casing registration feature 104. The X constraint and the XY constraint may be spaced apart from one another along the casing registration feature and positioned along an axis parallel to a Y axis of the base 101 and moveable stage (discussed further below).

[0119] In operation, the casing registration feature 104, equipped with an XY constraint 105 and X constraint 106 on its side surface facing the cassette 104a, is intended to limit the movement of the cassette casing 102 in the Y and X directions, respectively. A force may be applied to the length-wise (Y-direction) sides 102a, 103a of the cassette casing 102 by one of two different alignment mechanisms (described in further detail below) to push / engage the side of the cassette casing into the side of the casing registration feature facing the cassette casing 104a (as shown in FIG. 1A). The XY constraint 105 and X constraints 106 limit movement of the cassette casing by receiving at least one kinematic alignment feature disposed on an exterior side wall and projecting laterally outward (X-direction) of the cassette casing (best shown in Fig. 2).

[0120] In some embodiments, a presence sensor 107 may be located on the moveable stage 101 at a position where it may detect the presence of the cassette casing 102 on the chuck 109. A printed circuit board assembly (PCBA) 108, located on the XY stage,-16-FH12653613.3configured to control movement of the various components of the system may be actuated in response to feedback obtained from the presence sensor 107.

[0121] Assembly 200 - Casing And Casing Registration Feature

[0122] FIG. 2 depicts a top view of the casing registration feature 104, cassette casing, and kinematic alignment features in closer detail. As can be seen in FIG. 2, the cassette casing 102 may include at least one kinematic alignment feature 120 on the exterior side facing the casing registration feature 104. Each kinematic alignment feature 120 may be an extrusion from the exterior side of the cassette casing. Each kinematic alignment feature 120 may be spaced apart from the other in the Y- direction by a distance that may correspond the positions of the XY constraint 105 and X constraint 106 on the casing registration feature 104. Each of the kinematic alignment features 120 may include a rigid spherical portion.

[0123] In some embodiments, the at least one kinematic alignment feature includes a metal ball. In some embodiments, the at least one kinematic alignment feature includes a plastic ball.

[0124] In operation, the casing registration feature 104 is spaced apart from the cassette casing 102 until a force is received on the cassette casing 102 from the “A” direction (as shown in Fig. 2) by either a first or second alignment mechanism. When such force is provided to the unobstructed side 102a of the cassette casing (z.e., opposite of the casing registration feature 104), the kinematic alignment features 120 are pushed into or engaged with the XY and X constraint 105, 106. The X constraint 106 may be a substantially straight portion on the exterior side 104a of the casing registration feature. When the cassette casing 102 is pushed into the X constraint 106, a reactionary, normal force is produced in the opposite direction. The geometry of the X constraint 106 is configured to provide the normal force in the X direction, limiting movement in that direction. The XY constraint 105 may be a V-shaped groove, as discussed above. When the cassette casing 102 is pushed into the XY constraint 105, a reactionary, normal force is produced in the opposite X and Y directions, limiting movement in those directions.

[0125] In some embodiments, the casing registration feature may possess more than one XY constraint. In other embodiments, the casing registration feature may possess more than one X constraint.

[0126] Assembly 300 -Vacuum Chuck And Heat Transfer Device-17-FH12653613.3

[0127] FIG. 3A is front (X-direction) view of the imaging station assembly with chuck 109 and heat transfer device 130 in accordance with the present disclosure. FIG. 3B is a perspective exploded view of the chuck and heat transfer device in accordance with the present disclosure. When the chuck 109 is configured to apply vacuum pressure to the sample device and / or the cassette casing, the chuck 109 may be referred to herein as a vacuum chuck.

[0128] Referring to FIG. 3A, the cassette casing 102 is loaded on the vacuum chuck 109 (with the casing surfaces spaced from the chuck, and the substrate contained within the casing engaging the seating member 111 of the vacuum chuck 109) and secured there by a suctioning force. As discussed above, the vacuum chuck 109 may possess a “table-like” structure with a first leg 109a and second leg 109b. connected to a top surface 109c. Thus, an open space is defined between the first leg 109a, second leg 109b, and top surface 109c. The vacuum chuck 109 may include a heat transfer device. A heat transfer device 130 may be removably embedded in the space formed between the first leg 109a and second leg 109b of the vacuum chuck 109. As disclosed above, the heat transfer device 130 may be configured to keep the vacuum chuck 109 at a certain temperature. The heat transfer device 130 may comprise a thermoelectric (TEC) heat sink 110 and heating element / thermistor 118 in order to warm the sample while dissipating any excess heat. The heat sink 110 may be coupled to rest on the base 101 of the imaging station 100 and allows for the heating element 118 to rest on top of its upper surface. The upper surface of the heating element 118 contacts the lower surface of the top surface 109c of the vacuum chuck, warming it by conductive heat transfer.

[0129] In accordance with an aspect of the disclosure, as the substrate (containing the biological sample) is disposed in direct contact with the top surface of the seating member 111, the heat transfer device 130 disposed underneath the top surface of the seating member 111 provides direct heat transfer (via conduction) to the substrate (and biological sample). This can be advantageous as it both reduces the amount of thermal load required to achieve the desired temperature of the sample, and reduces the time required to reach the desired temperature than alternative heating configurations (e.g. applying heat through lids and / or other casing structures).

[0130] Referring to FIG. 3B, the heat sink 110 may be a substantially rectangularly shaped element. The heat sink 110 may include an elevated central portion 110b also possessing a substantially rectangular shape. The elevated central portion may include -18-FH12653613.3chamfered or filleted edges 110c at the width- wise sides to prevent rubbing and wear with the interior sides of the legs 109a, 109b, of the vacuum chuck 109. Apertures / holes IlOe may be formed at the corners of the elevated central portion 110b. The vacuum chuck 109 may include holes 109e at the corresponding location on the top portion 109c. These may be used to secure the heat sink 110 to the surface of the base 101 and vacuum chuck 109 by means of any suitable fastener (e.g. screw, bolt, rivet).

[0131] In some embodiments the heat sink 110 may be secured to the surface of the base 101 by magnetic coupling, adhesive bonding, or welding, depending on a user’s preferences.

[0132] A depression 110a may be formed at a central portion of the elevated central portion of the heat sink. The size and shape of the depression 110a may correspond to the size and shape of the heating element 118. The depression 110 may include “tab cut-outs” HOd at each comer. Each tab cut-out may include an aperture / hole 1 lOf that corresponds to a hole 109f on the surface of the vacuum chuck for receiving a suitable fastener for securing the heating element 118 between the lower surface of the vacuum chuck’s top portion 109c and the depression 110a of the heat sink 110.

[0133] In some embodiments the heating element 118 may be secured to the surface of the depression 110a by magnetic coupling, adhesive bonding, or welding, depending on a user’s preferences.

[0134] The corners of the top portion 109c of the vacuum chuck 109 may each also include an aperture 112 extending through the length of the first leg 109a and the second leg 109b. The apertures may be adaptable to receive any suitable fastener 121 (e.g. screw, bolt, rivet) to secure the first leg 109a and second leg 109b to the base 101 of the imaging station 100. In some embodiments, the vacuum chuck may be secured to the base 101 by magnetic coupling, adhesive bonding, or welding, depending on a user’s preferences.

[0135] The vacuum chuck 109 may include a vacuum port 113 connected into a side surface. The vacuum port 113 may include connection to a vacuum hose and suction generating device (not shown). In some embodiments, the suction generating device may be a vacuum pump or a fan. In some embodiments, the vacuum pump may be the GTEK Automation M00400-40E-A-24 Brushless DC Motor pump. In some embodiments, any suitable suction generating device candidate may be used in the imaging station assembly.-19-FH12653613.3

[0136] The top surface of the vacuum chuck 109 may include a seating member 111 in a substantially rectangular shape. The seating member 111 may be adapted to fit into a corresponding aperture on the lower surface of the base of the cassette casing. The seating member 111 may include at least one fluidic channel Illa. The fluidic channel Illa may be one continuous channel extending around the perimeter of the seating member 111. In some embodiments, the fluidic channels Illa may be any number of holes extending from the top of the seating member 111 downwards. In some embodiments, the fluidic channels Illa may be a plurality of disconnected, elongated channels extending across the surface of the seating member 111 in any preferred pattern.

[0137] In operation, when a cassette casing is placed onto the surface of the vacuum chuck 109, the aperture in the base surface of the cassette casing may engage with the seating member 111 on the vacuum chuck 109. Once the two components are aligned, the suction generating device may be actuated. A suctioning force extending through the suction hose and through the fluidic channel Illa may be established. The suctioning force may secure the base of the cassette casing with the surface of the vacuum chuck 109, preventing slippage and reducing the impact of unintended forces to the cassette casing.

[0138] Assembly 400 - First Exemplary Alignment Mechanism

[0139] FIG. 4A, FIG. 4B, and FIG. 4C are views of an exemplary imaging station assembly 100 holding a cassette casing 102 with a first exemplary alignment mechanism, referred to as an “off-stage alignment mechanism” 201 herein. Referring to FIG. 4A, a linear (in both X-Direction and / or Y-direction) stage drive 210 is configured to hold the movable stage 101, which supports a plurality of elements (as described above in connection with FIG.1 - FIG. 3B) on its surface. The XY moveable stage 101 is located on a stationary frame 203. An imaging device 206 may be located at an elevated position above (i.e. spaced in the Z-direction) the moveable stage 101, and moved relative the casing for image capture of the sample enclosed in the cassette casing 102 such that the imaging device may be at least partially disposed within the casing (i.e. the bottom of the objective lens can be disposed lower in the Z-direction than the upper surface of the cassette casing 102).

[0140] In operation, the linear drive 210 can be connected to an actuator 204 (e.g., a lead screw driven by a motor) which can automatically or manually be adjusted to vary the position of the stage in the X and Y direction. Additionally, the linear drive 210 can be coupled to a height adjustment mechanism 205 which may be for example, but not limited to -20-FH12653613.3a linear rail system. The height adjustment mechanism may guide the motion of the stage and cassette casing in the Z direction to align the sample substrate with an imaging device 206.

[0141] Referring to FIG. 4A, FIG. 4B, and FIG. 4C, the off-stage alignment mechanism 201 may be L-shaped and include a portion extending from the surface of the frame in a vertical (Z direction) 201b. A second portion 201a extending in the X and / or Y direction beyond the side of the XY stage and towards the exterior side 102a, 103a of the cassette casing 102 may be connected to the first portion 201b. Each portion of the off-stage alignment mechanism 201 may include a cut out (e.g. triangular shaped) such that the alignment mechanism has a plurality of trusses. These may reduce the weight of each offstage alignment portion while maintaining their structural stability. The distal free-end of the second portion of the off-stage alignment mechanism may include ball-plunger 207 aligned perpendicular to the at least one cassette registration feature 104. In another example, and without limitation the distal free end of the second portion may include a threaded spring plunger, press fit spring plunger, push pin spring plunger.

[0142] In accordance with an aspect of the disclosure, the off-stage alignment mechanism provides several advantages to alternative “on-platform” alignment mechanisms (e.g. mechanisms that are mounted on, and move with, the moveable stage 101). For example, configuring the alignment mechanism 201 as a fixed member, that is separate and apart from the moveable stage 101, avoids generating any vibrations associated with actuating and / or moving the alignment mechanism 201 - thereby providing more accurate placement of the casing, and thus more accurate image capture from the imaging device inserted therein.

[0143] In some embodiments, the off-stage alignment mechanism 201 may be removably mounted to the frame 203. The frame 203 may include a plurality of apertures 202a, 202b, for use with the alignment mechanism. The base of the first portion of the alignment mechanism 202 may possess a flat, open surface. An aperture may be formed on said surface configured to receive a suitable fastener (e.g. nut, bolt, screw, rivet) to secure the first portion 201b to the apertures in the frame 202a, 202b.

[0144] In some embodiments, the off-stage alignment mechanism may be fixed at a position adjacent to an exterior side of the XY stage 101. In some embodiments, the off-stage alignment mechanism 201 may be joined to the frame 203 through any suitable joining mechanism, such as welding or adhesive bonding.-21-FH12653613.3

[0145] In some embodiments, the first and second portions 201a, 201b of the offstage alignment mechanism may be removable from one another. Such a feature may facilitate easy removal for repair, transportation, or cleaning. In other embodiments, the first and second portions 201a, 201b of the off-stage alignment mechanism may form one continuous component.

[0146] In operation, a cassette casing 102 is loaded onto the surface of the vacuum chuck 109. The vacuum chuck 109 may apply a first vacuum pressure to the cassette casing 102. In some embodiments, the first vacuum pressure is about -0.1 inHG to about -15 inHg. In some embodiments, the first vacuum pressure is about -0.1 inHG to about -12.5 inHg. In some embodiments, the first vacuum pressure is about -0.1 inHG to about -10 inHg. In some embodiments, the first vacuum pressure is about -0.1 inHG to about -15 inHg. In some embodiments, the first vacuum pressure is about -0.1 inHG to about -7.5 inHg. In some embodiments, the first vacuum pressure is about -5 inHG to about -15 inHg. In some embodiments, the first vacuum pressure is about -5 inHG to about -12.5 inHg. In some embodiments, the first vacuum pressure is about -5 inHG to about -10 inHg. In some embodiments, the first vacuum pressure is about -5 inHG to about -7.5 inHg. In some embodiments, the first vacuum pressure is less vacuum than -15 inHG. In some embodiments, the first vacuum pressure is less vacuum than -10 inHG. In some embodiments, the first vacuum pressure is less vacuum than -5 inHG. When the cassette casing 102 is disposed on the surface of the chuck 109, its presence is detected by the presence sensor 107. Following the detection of the cassette casing 102 on the vacuum chuck, the linear drive 210 may move the moveable stage 100 from a first position to a second position where the cassette casing 102 is engaged with the alignment mechanism 201, and thereafter, the casing registration feature 104.

[0147] Referring to FIG. 4B, the XY stage may move along an actuator system 204 comprised of links. Each link 204a may form a flexible joint with another link, allowing for movement of the XY stage along an X direction and a Y direction as well. The stage may be moved in the Z direction to meet the height of the ball plunger 207 such that the plunger corresponds to the midpoint of the exterior side 102a, 103a of the cassette casing 102. The XY stage may be moved in the Z direction by the height adjustment mechanism 205, which may be a linear rail system.-22-FH12653613.3

[0148] The XY stage 100 moves to a position wherein the cassette casing 102 is perpendicular to ball plunger 207 of the off-stage alignment mechanism 201, with the ball plunger 207 brought into contact with the exterior side 102a, 103a of the cassette casing 102 to apply a force in the X-direction against the (right hand side as shown in Figu 4B) the casing. The first vacuum pressure applied by the chuck 109 may be configured to maintain the substrate in a substantially fixed location with respect to the Z-axis (i.e. parallel orientation with an XY plane defined by an upper surface of a seating member 111 of the chuck 109) while permitting the casing 102 to shift or move in the X-direction and / or Y-direction.

[0149] The off-stage alignment mechanism and ball-plunger 207 may remain fixed with respect to the frame 203, providing a reactionary, normal force onto an exterior side 102a, 103a of the cassette casing 102. This produced normal force engages the kinematic alignment features of the cassette casing 102 with the XY and X constraints of the casing registration feature 104, holding the casing securely between the end of the ball plunger 207 and the side 104a of the casing registration feature. Once the casing 102 is secured in the fully engaged position, i.e. with the kinematic alignment features 120 on the first side of the casing registered in the X constraint 106 and XY constraint 105, and the ball plunger 207 engaged on the opposite side of the casing, a second (e.g. higher) vacuum pressure can be applied on the seating member 111 to fixedly hold the casing in the Z-direction. In some embodiments, the second vacuum pressure is about -10 inHG to about -60 inHg. In some embodiments, the second vacuum pressure is about -10 inHG to about -50 inHg. In some embodiments, the second vacuum pressure is about -10 inHG to about -40 inHg. In some embodiments, the second vacuum pressure is about -10 inHG to about -30 inHg. In some embodiments, the second vacuum pressure is about -10 inHG to about -25 inHg. In some embodiments, the second vacuum pressure is about -10 inHG to about -21 inHg. In some embodiments, the second vacuum pressure is about -10 inHG to about -20 inHg. In some embodiments, the second vacuum pressure is about -10 inHG to about -15 inHg. In some embodiments, the second vacuum pressure is about -15 inHG to about -30 inHg. In some embodiments, the second vacuum pressure is about -15 inHG to about -25 inHg. In some embodiments, the second vacuum pressure is about -15 inHG to about -20 inHg. In some embodiments, the second vacuum pressure is a higher vacuum than about -15 inHG. In some embodiments, the second vacuum pressure is a higher vacuum than about -20 inHG. In some -23-FH12653613.3embodiments, the second vacuum pressure is a higher vacuum than about -25 inHG. In some embodiments, the second vacuum pressure is a higher vacuum than about -30 inHG.

[0150] Assembly 500- Second Exemplary Alignment Mechanism

[0151] FIGS. 5A - 5C depict a second alternative alignment mechanism which may be utilized in an exemplary imaging station. FIG. 5A depicts a view of the imaging station and second alignment mechanism. FIG. 5B depicts a side view of the imaging station and second alignment mechanism. FIG. 5C depicts a partial top view of the imaging station and second alignment mechanism. The second alignment mechanism will be referred to as the “on-stage alignment mechanism” 300 herein, as this alignment mechanism is mounted on the moveable stage 101 along with the chuck 109, casing registration feature 104.and casing 102 (when deposited from the forklift delivery device described herein).

[0152] Referring to FIG. 5A, the imaging station may include an on-stage alignment mechanism located on the XY movable stage 101. The on-stage alignment mechanism 300 may comprise an actuator 301 spaced apart from the exterior side 102a of the cassette casing 102. In some embodiments, the actuator 301 may be a stepper motor. In some embodiments, the actuator 301 may be a pneumatic actuator. In some embodiments, the actuator 301 may be an air cylinder. The actuator 301 may be secured to the base 101 by a mount 307. The mount 307 may comprise a set of tabs, with a horizontally oriented portion accommodating the shape of the bottom surface of the actuator 301 and the surface of the base 101. The horizontal tab may comprise apertures for fasteners (e.g. bolt, screw, rivet, nut) to secure the mount 307 to the base 101. The mount 307 may comprise a portion extending upwards and perpendicularly from the horizontal tab to conform to a side wall of the actuator. The upwards portion may comprise a hole for the actuator shaft to extend through. The upwards portion may comprise apertures located at each corner for any suitable fastener to fix the actuator to the mount 307. For example, and without limitation, the actuator 301 may also be secured to the base through any other suitable joining mechanism, such as welding or adhesive bonding.

[0153] The actuator shaft may be connected a rotatable cam. The rotatable cam may comprise a coupling 304 which may fit over the actuator shaft at one end and into a horizontal arm 302 at another. The horizontal arm 302 and vertical arm 303 are joined together at cam pin 306, which allows for movement of each arm in the horizontal and vertical directions, respectively. The horizontal arm 302 may fit into the actuator coupling -24-FH12653613.3204 at one end and join with the vertical arm 303 at another end by a cam pin 306. A second end of the vertical arm 303 may be mo veably joined with the actuator mount 307 by another pin or any other suitable mechanism. The end of the vertical arm 303 where the cam pin 306 is secured may include a protrusion 305 configured to contact the exterior side 102a, 103a of the cassette casing 102 at its midpoint.

[0154] In some embodiments, the protrusion 305 may be shaped like a finger with a portion extending outwards in the horizontal (X-direction) from the vertical (Z-direction) arm 303. As shown in the exemplary embodiment of FIG. 5B, the finger 305 can engage the surface of the casing 102 when the arm 303 is aligned, or oriented generally parallel, with the side of the casing 102.

[0155] In other embodiments, the protrusion 205 may comprise any suitable shape for contacting the exterior side 102a, 103a of the cassette casing 102. For example, and without limitation, the protrusion 205 may be a lip, flange, or filleted edge.

[0156] In operation, when a cassette casing 102 is initially loaded onto the chuck 109, with the casing is spaced from the casing registration feature 104 at a first position. In some embodiments, the chuck 109 may apply a first vacuum pressure to the substrate, as described above to maintain the casing in a level position (i.e., parallel to the planar upper surface of the seating member 111 of the chuck). In some embodiments, the chuck 109 does not have vacuum capability. After the cassette casing 102 is disposed on the chuck 109, the actuator 301 may be automatically actuated through the feedback relayed from the presence sensor 107. In some embodiments, the actuator 301 may be manually actuated. When the actuator 301 is actuated, the actuator shaft and coupling 304 are rotated, consequently rotating the cam assembly, 302, 303, 305 (i.e., the horizontal arm, vertical arm, and protrusion).

[0157] Referring to FIG. 5B and FIG. 5C, as the cam assembly is rotated, the protrusion 305 is moved forwards into a position where the edge of protrusion 305 contacts the exterior side 102a, 103a of the cassette casing 102. A second force is exerted by the cam assembly and protrusion 305 onto the cassette casing 102 and may push / engage the casing into the casing registration feature 104 into a second position. The second force may be configured to maintain the substrate in a substantially parallel orientation with an XY plane defined by an upper surface of the chuck 109. As shown in FIG. 5C, the cassette casing 102 includes a kinematic registration feature 120 which is engaged with the XY constraint 105 to stabilize the cassette casing 102. Combined with the suctioning force on the substrate within -25-FH12653613.3the cassette casing 102 from the chuck 109, the alignment mechanism 300 secures the cassette casing into a position where it can be imaged by an imaging device in a precise and predetermined location, thereby providing optimal image quality.

[0158] Again, the pressure applied by the chuck can vary from an initial / first pressure which maintains the substrate (and cassette casing) in a flat orientation parallel to the seating member 111, but permitting XY movement of the cassette casing, to a second (e.g. higher) pressure once the cam 305 and registration feature 104 are also engaged with the cassette casing. The first pressure can provide a balancing force to counteract any rotation moment induced by an offset center of gravity of the cassette casing 102 (when mounted on the chuck 109).

[0159] Assembly 600 - Cassette Lifting Mechanism

[0160] FIG. 6 A and FIG. 6B are views of the cassette casing and transfer mechanism loading and unloading the cassette casing into the imaging station in accordance with the present disclosure. The lifting mechanism 501 can be configured to engage the cassette casing 102. As shown in FIGS. 6A and FIG. 6B, the lifting mechanism 501 includes an arm base 504 and parallel arms 510a, 510b extending from the arm base in a perpendicular direction. The arm base 504 can include a body (e.g., a rectangular body) having a top surface, a bottom surface and multiple sides, with the arms 510a, 510b extending from a side of the arm body. In some embodiments, the arm base 504 has a boss feature 511 (z.e., a raised portion on the surface of the body) extending from the top surface of the arm base body. In some embodiments, the boss features may be a raised rectangular portion or any other preferred shape. In other embodiments, the boss feature may be a pin or a shaft. A locating pin 512 (e.g., cylindrical or rectangular pin) having a first end, a second end, and a thickness therebetween can be disposed on the top surface of the arm base 504, with the longitudinal axis of the locating pin 512 oriented perpendicular to the top surface. The locating pin 512 is sized and shaped to receive a locating aperture of a corresponding shape on the underside of the base portion 103 of the cassette casing 102.

[0161] In some embodiments, the first arm 510a and second arm 510b can be configured in a manner akin to tines of a forklift to engage and lift a cassette casing 102 (e.g., from the bottom of the assembly). In some embodiments, the first arm 510a and second arm 510b are substantially rectangular. In some embodiments, the first arm 510a and second arm 510b are of a length corresponding to the length of the cassette casing base 103. In some -26-FH12653613.3embodiments, the top surface of at least one arm of the lifting mechanism 510a, 510b includes a boss feature 51 la, 51 lb. For example and without limitation, the top surface of the first arm 510a can include a boss feature 511a (z.e., a raised portion on the top surface), and the top surface of the second arm 510b can include a boss feature 511b (e.g., as shown in FIG. 6B). The boss features 511a, 511b extend perpendicularly from the top surface of the arms 510a, 510b respectively, and are positioned and shaped to correspond to the apertures in the base of the cassette casing 102. In some embodiments, the boss features 511a, 511b have the same geometry while in others they do not. In some embodiments, the boss features 511a, 511b are laterally aligned at the same position along the first arm 510a and second arm 510b.

[0162] In some embodiments, the lifting mechanism 501 can include a recessed portion 512 defining an interior portion configured to receive at least a portion of the assembly.

[0163] In operation, the lifting mechanism 501 can be moved (e.g., laterally, vertically and / rotationally) into alignment with the assembly. In some embodiments, the lifting mechanism 501 is attached to an instrument gantry. The lifting mechanism 501 can be maneuvered beneath the assembly, positioning the arms 510a, 510b to align the boss features 511a, 511b of the lifting mechanism 502 with the registration features of the assembly. Once aligned, the lifting mechanism 502 can be raised to engage and lift the assembly. The spacing between the first arm 510a and the second arm 510b is such that when the lifting mechanism 501 is engaged with the assembly, outer sides of the lifting mechanism 501 are coplanar with exterior sides of the cassette casing 102 as shown in FIG. 6A.

[0164] Assembly 700 - Cassette Lifting Mechanism

[0165] FIG. 7 A depicts a partial view of the imaging station assembly and loading station, holding multiple cassette casing assemblies, with an off-stage alignment mechanism in accordance with the present disclosure. The loading station 410 can be configured to hold any variation of the exemplary cassette casing 102. In the exemplary embodiment illustrated, a plurality of bays 405 are provided on the loading station, each configured to receive a cassette casing 102. Each bay 405 may be a rectangularly- shaped enclosure configured to support the bottom surface of the cassette casing 102. In some embodiments, the bay 405 is configured to receive an aperture at the cassette casing base 103 and contacts the substrate / sample. Each bay can include a pin 408 configured to receive a first locating aperture of the cassette casing assembly 102. The pin 408 can be disposed at one of end of -27-FH12653613.3the bay, with its longitudinal axis perpendicular to the surface of the loading station. In some embodiments, the pin 408 may be cylindrical, rectangular, or include a locking mechanism.

[0166] In operation, a lifting mechanism 501 can lift an assembly and position it onto a bay 405. The lifting mechanism 501 can transfer the cassette casing 102 between different bays 405 or can transfer it between different stations (e.g., the loading station, an imaging station, a fluidics station). As the assembly is lowered onto the bay 405, the pin 408 extends through the first locating aperture of the cassette casing base 103. In some aspects, this can align and / or secure the assembly 102 on the bay 405.

[0167] The transfer mechanism 501 may be connected to an elevator mechanism 400 and rail system 407. The elevator mechanism 400 may be configured to move the transfer mechanism in the Z direction and may comprise a coupling component 404 to engage with an extrusion on the arm base 509 of the transfer mechanism. The coupling component 404 may be of a substantially square shape, with a top and bottom surface, defining a thickness therein. The coupling component may include an aperture at the bottom surface shaped to correspond to an extrusion of the arm base 504. In some embodiments, the coupling component 404 may include a magnet disposed at the surface to be engaged with the arm base 504. The arm base may include a corresponding magnet, such that a magnetic force is established between the arm base and coupling component 404. The coupling component 404 may rest on an elevator mechanism 400, which may include a microcontroller 406, lifting shaft 402, and conveyor belt 401. A lifting shaft 402 may be of a substantially rectangular shape and may be disposed along a vertical axis. It may possess a seat 403, a portion extruding outwards from the lower edge of the lifting shaft 402 with a width corresponding to that of the shaft. The seat 403 may support the coupling component 404. The lower surface of the seat 403 may include an aperture corresponding to that of the extrusion on the arm base and the aperture on the lower surface of the coupling component 404. In some embodiments, the seat 403 may be thin enough such that a magnetic force is established and maintained between the lower surface of the coupling component 404 and the lifting mechanism 501. The seat 403 may be coupled to the lifting shaft 402 by a hinge assembly (z.e. a pin and leaves or knuckles). The lifting shaft 402 may include a sleeve at the upper portion of the side wall wherein a conveyer belt loop 401, oriented in a vertical direction, may be fed through. The entire elevator mechanism may be mo veably joined to a rail system 407. The rail system 407 may comprise a series of links-28-FH12653613.3joined to one another on which the elevator mechanism 400 may move along in the X and Y directions.

[0168] In operation, the lifting mechanism 501 may be moved by the elevator mechanism 400 and the rail system 407 to a position above the bay 405 and a cassette casing 102. When the lifting mechanism 501 is aligned with an elevator mechanism 400 (z.e. the aperture in the seat 403 of the lifting shaft 402 and the lower surface of the coupling mechanism 404 is aligned with the protrusion on the arm base of the lifting mechanism 501), the elevator mechanism 400 may be controlled to move upwards or downwards in a vertical direction. As the conveyor belt 401 coupled to the sleeve of the lifting shaft 402 is actuated, the belt 401 moves in the vertical direction, consequently moving the lifting shaft 402, coupling component 404, and lifting mechanism 501 as well. After the desired height is achieved, the elevator mechanism 400 and lifting mechanism 501 may be moved in the X and Y directions along a rail system 407. The movement of the elevator mechanism 400 and rail system 407 may be driven by connection to a motor, air cylinder, electric cylinder, etc. (not shown). The movement of coupling component 404 along the elevator mechanism 400 and rail system 407 may be further controlled by instructions provided by the microcontroller 406.

[0169] Assembly 800

[0170] FIG. 7B and FIG. 7C are side views of the imaging station 100 in accordance with the present disclosure. Referring to FIG. 7B, a first recess 117a and second recess 117b may be formed at opposing sides between the chuck 109 and heat transfer device 110, and the cassette casing 102 configured to receive tines of a transfer mechanism / forklift. The recesses 117a, 117b formed may be used as an anchor for a lifting mechanism. The lifting mechanism can be used to engage the cassette casing 102 to align it onto the surface of the chuck 109 when beginning an imaging process. Further, at the end of an imaging process, the lifting mechanism may be used to remove the cassette casing from the imaging station assembly. The tines of the forklift / lifting mechanism 501 may extend outwards to into the recesses 117a, 117b formed by the lower exterior surface of the cassette casing 102, the chuck 109, and heat transfer device 110 to lift and move the cassette casing 102 to a desired location.

[0171] FIG. 7B and FIG. 7C show a clearance cut 208 out in an imaging device 206. The clearance cut out 208 may be L-shaped and provide space for loading and unloading the-29-FH12653613.3cassette casing 102 from the chuck 109 without obstruction. In some embodiments, the clearance cut out 208 may be arc shaped.

[0172] In some embodiments, the clearance cut out 208 may provide about 23 mm of space in the vertical (Z-direction) between the upper surface of the cassette casing 102 and the bottom surface of the imaging device objective lens - for the cassette casing 102 to be loaded and unloaded into the imaging station assembly 100.

[0173] Referring to FIG. 7C, the removal of the cassette casing from the imaging station assembly in operation is depicted. In some embodiments, only about 8 mm of space in the vertical direction is required to remove the cassette casing.

[0174] In operation, when a cassette casing 102 is raised from or lowered onto the chuck 109. In some embodiments, prior to moving the cassette casing, the imaging device 206 is translated horizontally in an opposite direction to prevent interference between the cassette casing and the imaging device lens 206a. While a predefined clearance (e.g., 8mm of space) in the vertical direction is required for the cassette casing 102 to be removed from the imaging station assembly 100, the clearance cut out 208 may provide up to a larger clearance amount (e.g., 23 mm of space) in the vertical direction. In some embodiments, the dimensions of the clearance cut out 208 may be adjustable depending on the dimensions of the other components in the system. In some embodiments, the dimensions of the cassette casing 102 components and the imaging station 100 may be adjustable such that more or less than 8 mm is required to insert or remove the cassette casing 102 into the imaging station 100.

[0175] Assembly 900

[0176] FIG. 8A is a top view of the printed circuit board. FIG. 8B is a top view of the cable routing of the exemplary imaging station assembly.

[0177] Referring to FIG. 8A the printed circuit board assembly (PCBA) 108 may comprise a computer-readable storage medium with program instructions embodied therewith. The PCBA may comprise program instructions for actuating and controlling the operation of the imaging station assembly and may be selected from any suitable controller or microcontroller. A port for each component to be controlled within the imaging station 100 may be configured on the PCBA. For example, and without limitation, the PCBA may include ports for a stepper motor, TEC heat sink, thermistor / heating element, presence sensor, and an FMC-ISB ribbon.-30-FH12653613.3

[0178] Referring to FIG. 8B, the ports included in the PCBA may be connected to each respective component. For example, the stepper motor port may be connected to the stepper motor in an embodiment of the disclosed subject matter which includes a stepper motor alignment mechanism. The thermistor port may be connected to the thermistor / heating element. The presence sensor port may be connected to the presence sensor and so on.

[0179] In operation and in the context of the disclosed subject matter, when the presence sensor detects the presence of a cassette casing on the chuck feedback is relayed to the PCBA. The PCBA may then then send instructions to the vacuum chuck to actuate a suction force to hold the cassette casing in place. Further, instructions may be sent by the PCBA to the thermistor / heating element and TEC / heat sink to warm the vacuum chuck and dissipate excess heat.

[0180] In some embodiments, the PCBA may be configured to actuate the movement of the on-stage alignment mechanism.

[0181] In some embodiments, the PCBA may be configured to actuate and control the movement of the XY stage and off-stage alignment mechanism.

[0182] Assembly 1000 and 1100

[0183] FIG. 9 - FIG. 10 are top views of an exemplary fluidic station 1000 and 1100 holding an assembly 100. The fluidics station 1000, 1100 can be configured to hold various assemblies depending on a processing method to be carried out on a sample. The fluidics station 1000, 1100 can include a base 220, an XY stage 210 (e.g., a moveable stage) coupled to the base, and a removable lid pivotably coupled to the base (not shown). The stage 210 can be connected to an actuator (e.g., a lead screw driven by a motor) which can automatically or manually be adjusted to vary the position of the stage 210. Additionally, the stage can be coupled to a linear rail system which guides the motion of the stage when it is raised or lowered. The stage 210 can include a casing registration feature 104 and chuck (not shown), which is configured to align and secure the imaging station assembly 100 on the fluidics station 1000.

[0184] Referring to FIG. 9, the lifting mechanism (not shown) can be used to dispose the cassette casing 102 onto the surface of the vacuum chuck (not shown). The imaging station assembly may include a casing registration feature 104 and one of the two exemplary casing alignment mechanisms configured to align the imaging assembly 100. The casing alignment mechanism can include an off-stage alignment mechanism 201 and one or more -31-FH12653613.3casing registration features 104. The casing registration features 1010 can be rectangular bodies having an exterior surface configured to engage the kinematic alignment features (e.g., kinematic alignment features 120) of the cassette casing 102. When the cassette casing 102 is positioned on the vacuum chuck, the casing alignment mechanism can be activated. The XY stage 210 may be actuated to translate in the X and Y direction, contacting the ball plunger 207 of the off-stage alignment mechanism 201. The translation of the XY stage 210, and consequently cassette casing 102, into contact with the ball plunger 207, can apply a force on an exterior side of the cassette casing 102, pushing it against the casing registration features 104 and engaging the feature with the kinematic alignment features 120 of the assembly (e.g., as shown in FIG. 9).

[0185] Referring to FIG. 10, the lifting mechanism (not shown) can be used to engage the cassette casing with the vacuum chuck (not shown). The imaging station can further include a casing registration feature 104 and one of the two exemplary casing alignment mechanisms configured to align the cassette casing 102. The casing alignment mechanism can include an on-stage alignment mechanism 300 and one or more casing registration features 104. In some embodiments, the casing alignment mechanism is a cam driven by an actuator (e.g., a stepper motor as shown in FIG. 10, or any suitable alternative). The casing registration feature 104 can be an elongate body having an exterior surface configured to engage the kinematic alignment features 120 of the cassette casing assembly 102.

[0186] When the cassette casing assembly 102 is positioned on the vacuum chuck (not shown), the casing alignment mechanism 300 can be (e.g., manually or automatically) activated. In some embodiments, the cam assembly 302-306 is configured to rotate to contact the assembly. The protrusion 305 of the cam assembly can apply a force on an exterior side of the cassette casing, pushing it against the casing registration feature 104, and thus engaging the casing feature 104 with the kinematic alignment features 120 of the cassette casing 102 (e.g., as shown in FIG. 10).

[0187] Assembly 1200

[0188] FIG. 11 depicts the presence sensor of the imaging station assembly a non-exhaustive list of suitable presence sensor candidates. Exemplary presence sensors include the Panasonic GX-F15A, Panasonic GX-F8A, Autonics PSB40-20DN, and TT Electronic s / Optek OPB732WZ sensor. This list is not exhaustive, and any suitable presence sensor candidate may be used in the imaging station assembly.-32-FH12653613.3

[0189] In some preferred embodiments, a reflective based presence sensor may be used due to their decreased propensity to falsely detect components in the assembly other than the cassette casing.

[0190] In other embodiments, an inductive presence sensor may be used.

[0191] FIG. 12 depicts a pedestal 1209 (which may be referred to as a “chuck”) having a three-point contact mechanism 121 la-1211c for aligning and / or registering a sample device 114 (e.g., a cassette) to an imaging station. In some embodiments, the pedestal 1209 includes a first contact mechanism 1211a, a second contact mechanism 1211b, and a third contact mechanism 1211c arranged such that the sample device 114 is fully constrained (z.e., motion is restricted for all degrees of freedom). As shown in FIG. 12, in some embodiments, each contact mechanism 121 la- 1211c includes a spherical contact mounted on a pillar. For example, the spherical contact may be a ball bearing (e.g., stainless steel ball bearing, ceramic ball bearing, etc.). In some embodiments, the pillars are integrally formed with the pedestal 1209. In some embodiments, the pillars are attached to the pedestal 1209 as separate components, e.g., using fixation members such as machine screws. As shown in FIG. 12, the sample device 114 includes corresponding receiving structures 114a-114c (e.g., recesses or apertures) for engaging the three-point contact mechanism 121 la-1211c. In some embodiments, each contact mechanism 121 la-1211c contacts the substrate (e.g., glass slide) secured within the sample device 114 (each contact mechanism 1211 a- 1211c extends through the bottom portion of the sample device on which the substrate is positioned). In some embodiments, each contact mechanism 121 la-1211c directly contacts the bottom portion of the sample device 114 (and does not contact the substrate at all).

[0192] Imaging Station 1300

[0193] FIG. 13A illustrates an imaging station 1300 having a moveable stage 1301 with a pedestal 1309 having magnetic components 1470a, 1470b for magnetically coupling magnetic components in a sample device (e.g., a cassette assembly). The pedestal 1309 is affixed to the moveable stage 1301 via a plurality of fixation members (e.g., screws) inserted within a plurality of through holes. In some embodiments, the pedestal 1309 is integrally formed with the moveable stage 1301 (e.g., via additive manufacturing). In some embodiments, the moveable stage 1301 includes a first linear stage configured for motion in an X direction and a second linear stage configured for motion in a Y direction. The image station 1300 includes a cassette presence sensor 1390 configured to detect the presence (or -33-FH12653613.3absence) of a sample device positioned on the pedestal 1309. The cassette presence sensor 1390 may include an optical sensor, such as an infrared sensor. Other suitable presence sensors are described in more detail above. The pedestal 1309 further includes a peripheral portion 1392 that is configured to receive a peripheral apparatus. In some embodiments, the peripheral apparatus is a hemispherical reference tool for calibrating an optical system (e.g., calibrating the z-motion stage and / or z-motion encoder). In some embodiments, the peripheral apparatus is an objective cleaning tool. In some embodiments, the peripheral apparatus is an optical alignment tool or optical calibration tool for aligning and / or calibrating the optical system.

[0194] FIGS. 13B-13C illustrates a pedestal 1309 having magnetic components for magnetically coupling magnetic components 1470a, 1470b in a sample device (e.g., a cassette assembly). In some embodiments, the moveable stage 1301 is positioned on a frame that is configured to support an optical train. For example, the frame may substantially encircle the moveable stage and support an optical bench plate on which an objective lens and other optical components (e.g., for an emission / detection pathway and an excitation / illumination pathway). The pedestal 1309 has at least one side, for example, a first side 1382a that is opposite a second side 1382b. In some embodiments, where the pedestal 1309 is rectangular in shape, the first side 1382a and the second side 1382b are long sides. In some embodiments, where the pedestal 1309 is rectangular in shape, the pedestal 1309 includes a first short side 1382c and a second short side 1382d. As shown in FIGS. 13A-13C, the first side 1382a includes a first recess 1384a and the second side 1382b includes a second recess 1384b. A first magnetic component 1470a is positioned (e.g., secured via adhesive) within the first recess 1384a and a second magnetic component 1470b is positioned (e.g., secured via adhesive) within the second recess 1384b. Because the first side 1384a and the second side 1384b are parallel, the first magnetic component 1470a and the second magnetic component 1470b are also parallel to one another. In some embodiments, one or more grooves are formed in the sides of the pedestal 1309 and intersecting the recesses 1384a, 1384b to aid in removal and / or replacement of the magnetic components 1470a, 1470b.

[0195] In some embodiments, the one or more magnetic components includes a ferromagnetic material. In some embodiments, the ferromagnetic material includes iron, alloy steel, stainless steel, nickel, cobalt, gadolinium, neodymium, ferromagnetic ceramic, or a combination thereof-34-FH12653613.3

[0196] The pedestal 1309 includes one or more seating member configured to contact the substrate and / or the sample device, such as seating members 131 la- 1311c. As shown in FIGS. 13A-13C, the seating members 1311 a- 1311c extend from a top surface of the pedestal 1309. The seating members 131 la- 1311c include a three-point support and are discontinuous about the top surface of the pedestal 1309. In some embodiments, the seating member is continuous (e.g., has a continuous planar surface extending) about a perimeter of the top surface of the pedestal 1309. In some embodiments, the pedestal 1309 includes a stepped configuration where the base of the pedestal 1309 is wider (or longer) than subsequent portions above the base of the pedestal 1309. Seating members 1311a and 1311b are generally positioned at the comers of the top surface of first short side 1382c and have smaller planar surfaces, while seating member 1311c has a larger planar surface (larger than seating members 1311a, 1311b) and extends along the top surface of second short side 1382d. In some embodiments, the top surface of one or both of first long side 1382a and second long side 1382b include one or more seating members (e.g., with any suitable size / area of the planar surfaces). In some embodiments, seating members are precision manufactured to be very flat and at precisely the same height (z.e., very small tolerances) as the other seating members. This can be accomplished by machining all seating members at the same time (e.g., without adjusting the part in the machine tool). As will be explained in more detail below, the pedestal 1309 may include substantially vertical sides. In some embodiments, the pedestal 1309 includes a chamfer between the side and the top surface that is configured to engage a complimentary angled feature in the base or bottom portion of the sample device (e.g., a cassette assembly).

[0197] FIGS. 14A-14C depict various cross-sectional views of systems 1400a-1400c including a sample device 1514 (e.g., a cassette assembly) having a base 1502 with magnetic components 1570a, 1570b positioned on a pedestal 1409 having corresponding magnetic components 1470a, 1470b. The sample device 1514 may be the same or similar as the sample devices described above, including sample device 114. In particular, the sample device 1514 (including a top 1504 releasably coupled to the base 1502 with substrate 1506 secured therebetween) may be positioned on a pedestal 1409 for imaging and / or fluidic operations to be performed on the one or more samples disposed within the open well of the sample device 1514. As shown in FIG. 14A, the pedestal 1409 includes sides 1482a, 1482b (which may be referred to as support walls) that are sized to engage (i.e., contact) the-35-FH12653613.3substrate (e.g., substrate 1506) and / or the sample device 1514 directly. In some embodiments, a width and / or length of the sides 1482a, 1482b is less than a width and / or length of the second opening 1533 (e.g., aperture) of the sample device 1514. In some embodiments, as shown in FIG. 14A, the magnetic components 1470a, 1470b in the pedestal 1409 that correspond to the magnetic components 1570a, 1570b in the base 1502 are secured within the pedestal 1409 (and extend parallel with the sides of the pedestal, and base, respectively). For example, the magnetic components 1470a, 1470b may be integrally formed within the pedestal 1409 or may be inserted into a bore formed in the sides 1482a, 1482b of the pedestal 1409. In another example, a first magnetic component 1470a may be inserted into a first recess 1484a formed in a first side 1482a of the support wall of the pedestal 1409 and a second magnetic component 1470b may be inserted into a second recess 1484b formed in a second side 1482b of the support wall. In some embodiments, the magnetic components 1470a, 1470b in the pedestal 1409 are a ferromagnetic material, such as neodymium (a neodymium magnet). In some embodiments, one or more of the magnetic components 1470a, 1470b, 1570a, 1570b are magnetized to generate a magnetic field. In some embodiments, the magnetic components 1470a, 1470b and / or the magnetic components 1570a, 1570b are electromagnets (e.g., generate a magnetic field when energized with electricity). When the sample device 1514 (including the base 1502 and the top 1504) is positioned on the pedestal 1409, the magnetic components 1470a, 1470b in the pedestal 1409 magnetically couple to the respective magnetic components 1570a, 1570b in the sample device 1514 with sufficient force to thereby secure the sample device 1514 to the pedestal 1409. In some embodiments, the magnetic force is sufficient to secure the sample device 1514 to the pedestal 1409, but still allows for removal of the sample device 1514 from the pedestal 1409, for example, by an automated / robotic transport arm or manually by hand.

[0198] FIGS. 14B and 14C shows a cross-sectional front view of systems 1400b, 1400c where the pedestal 1409 includes recessed 1484a, 1484b (e.g., side cutouts) configured to receive the magnetic components 1470a, 1470b (e.g., magnetic blocks). In FIG. 14B, the exterior surface of the sides 1482a, 1482b of the support walls of the pedestal 1409 are substantially vertical. One or more seating members 1411 extend from the top surface of the pedestal 1409 and are configured to contact the substrate 1506 and / or the sample device 1514. In FIG. 14C, the exterior surface of the sides 1482a, 1482b of the support walls 1482 of the pedestal 1409 includes chamfers 1482c, 1482d (e.g., angled sides) to substantially -36-FH12653613.3match an angle of at least a portion of the bottom surface of the base 1502 (extending to the perimeter of the second opening 1533). In some embodiments, the sample device 1514 is additionally secured to the pedestal (e.g., motion is reduced or prevented in the XY plane) when the exterior surface of the sides 1482a, 1482b of the support wall includes an angle that matches the angle of at least a portion of the bottom surface of the base 1502 (around the second opening 1533).

[0199] In some embodiments, as shown in FIG. 14C, one or more pusher elements 1450 are included on opposing sides to further support the sample device 1514. In some embodiments, the pusher elements 1450 are pads (e.g., polymer pads). In some embodiments, the pusher elements are plungers (e.g., spring-loaded plungers) configured to provide a reaction force against the bottom of the base 1502 of the sample device 1514 (e.g., in a direction opposite the pull of the magnetic force). In some embodiments, the pusher elements 1450 are used to decouple the sample device 1514 from the pedestal 1409 (e.g., provide a force to increase the distance between the magnetic components 1470a, 1470b and magnetic components 1570a, 1570b to thereby reduce the magnetic force therebetween).

[0200] The flow devices of the disclosure may include any suitable material, for example, polymeric materials, such as polyethylene or polyethylene derivatives, such as cyclic olefin copolymers (COC), polymethylmethacrylate (PMMA), polydimethylsiloxane (PDMS), polycarbonate, polystyrene, polypropylene, polyvinyl chloride, polytetrafluoroethylene, polyoxymethylene, polyether ether ketone, polycarbonate, polystyrene, or the like, or they may be fabricated in whole or in part from inorganic materials, such as silicon, or other silica based materials, e.g., glass, quartz, fused silica, borosilicate glass, metals, ceramics, and combinations thereof.

[0201] In some embodiments, the present devices may be assembled by alignment and stacking of the slide, tissue sample, fluidic interface layer, gasket, and adaptor. For example, the adaptor may be aligned with and placed onto the fluidic interface layer, or the adapter can be placed on the slide prior to dispensing the fluidic interface layer of reagent(s). Compression may be applied during assembly such that the fluidic interface layer, gasket, and / or substrate layer are reversibly attached. Assembly, and disassembly, can be performed manually.

[0202] Surface Properties-37-FH12653613.3

[0203] A surface of the device may include a material, coating, or surface texture that determines the physical properties of the device. In particular, the flow of liquids may be controlled by the surface properties (e.g., wettability of a liquid-contacting surface). In some cases, a portion (e.g., a flow path) may have a surface having a wettability suitable for facilitating liquid flow (e.g., in a flow path).

[0204] Wetting, which is the ability of a liquid to maintain contact with a solid surface, may be measured as a function of a water contact angle. A water contact angle of a material can be measured by any suitable method known in the art, such as the static sessile drop method, pendant drop method, dynamic sessile drop method, dynamic Wilhelmy method, single-fiber Wilhelmy method, single-fiber meniscus method, and Washburn’s equation capillary rise method.

[0205] For example, portions of the device carrying aqueous phases (e.g., a channel or flow path) may have a surface material or coating that is hydrophilic or more hydrophilic than the other parts of the device, e.g., include a material or coating having a water contact angle of less than or equal to about 90°, and / or other components of the device may have a surface material or coating that is hydrophobic or more hydrophobic than the flow path, e.g., include a material or coating having a water contact angle of greater than 70° (e.g., greater than 90°, greater than 95°, greater than 100° (e.g., 95°-120° or 100°-10°)). The system can be designed to have a single type of material or coating throughout. Surface textures may also be employed to control fluid flow.

[0206] The surface properties may be those of a native surface (i.e., the surface properties of the bulk material used for fabrication) or of a surface treatment. Non-limiting examples of surface treatments include, e.g., surface coatings and surface textures. In one approach, the surface properties are attributable to one or more surface coatings present in a portion. Hydrophobic coatings may include fluoropolymers (e.g., AQUAPEL® glass treatment), silanes, siloxanes, silicones, or other coatings known in the art. Other coatings include those vapor deposited from a precursor such as henicosyl-1,1,2,2-tetrahydrododecyldimethyltris(dimethylaminosilane) ; henicosyl- 1 , 1 ,2,2-tetrahydrododecyltrichlorosilane (Cl 2); heptadecafluoro- 1,1, 2,2-tetrahydrodecyltrichlorosilane (CIO); nonafluoro- 1,1, 2,2-tetrahydrohexyltris(dimethylamino)silane; 3,3,3,4,4,5,5,6,6-nonafluorohexyltrichlorosilane; tridecafluoro- 1 , 1 ,2,2-tetrahydrooctyltrichlorosilane (C8) ; bis (tridecafluoro- 1 ,1,2,2- -38-FH12653613.3tetrahydrooctyl)dimethylsiloxymethylchlorosilane; nonafluorohexyltriethoxysilane (C6); dodecyltrichlorosilane (DTS); dimethyldichloro silane (DDMS); or 10-undecenyltrichlorosilane (VI 1); pentafluorophenylpropyltrichlorosilane (C5). Hydrophilic coatings include polymers such as polysaccharides, polyethylene glycol, polyamines, and polycarboxylic acids. Hydrophilic surfaces may also be created by oxygen plasma treatment of certain materials.

[0207] A coated surface may be formed by depositing a metal oxide onto a surface of the system. Example metal oxides useful for coating surfaces include, but are not limited to, A12O3, TiO2, SiO2, or a combination thereof. Other metal oxides useful for surface modifications are known in the art. The metal oxide can be deposited onto a surface by standard deposition techniques, including, but not limited to, atomic layer deposition (ALD), physical vapor deposition (PVD), e.g., sputtering, chemical vapor deposition (CVD), or laser deposition. Other deposition techniques for coating surfaces, e.g., liquid-based deposition, are known in the art. For example, an atomic layer of A12O3 can be deposited on a surface by contacting it with trimethylaluminum (TMA) and water.

[0208] In another approach, the surface properties may be attributable to surface texture. For example, a surface may have a nanotexture, e.g., have a surface with nanometer surface features, such as cones or columns, that alters the wettability of the surface.Nanotextured surface may be hydrophilic, hydrophobic, or superhydrophobic, e.g., have a water contact angle greater than 150°. Exemplary superhydrophobic materials include Manganese Oxide Polystyrene (MnO2 / PS) nano-composite, Zinc Oxide Polystyrene (ZnO / PS) nano-composite, Precipitated Calcium Carbonate, Carbon nano-tube structures, and a silica nano-coating. Superhydrophobic coatings may also include a low surface energy material (e.g., an inherently hydrophobic material) and a surface roughness (e.g., using laser ablation techniques, plasma etching techniques, or lithographic techniques in which a material is etched through apertures in a patterned mask). Examples of low surface energy materials include fluorocarbon materials, e.g., polytetrafluoroethylene (PTFE), fluorinated ethylene propylene (FEP), ethylene tetrafluoroethylene (ETFE), ethylene chlorotrifluoroethylene (ECTFE), perfluoro-alkoxyalkane (PFA), poly(chloro-trifluoro-ethylene) (CTFE), perfluoro-alkoxyalkane (PFA), and poly (vinylidene fluoride) (PVDF). Other superhydrophobic surfaces are known in the art.-39-FH12653613.3

[0209] In some cases, the water contact angle of a hydrophilic or more hydrophilic material or coating is less than or equal to about 90°, e.g., less than 80°, 70°, 60°, 50°, 40°, 30°, 20°, or 10°, e.g., 90°, 85°, 80°, 75°, 70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15°, 10°, 9°, 8°, 7°, 6°, 5°, 4°, 3°, 2°, 1°, or 0°. In some cases, the water contact angle of a hydrophobic or more hydrophobic material or coating is at least 70°, e.g., at least 80°, at least 85°, at least 90°, at least 95°, or at least 100° {e.g., about 100°, 101°, 102°, 103°, 104°, 105°, 106°, 107°, 108°, 109°, 110°, 115°, 120°, 125°, 130°, 135°, 140°, 145°, or about) 150°.

[0210] The difference in water contact angles between that of a hydrophilic or more hydrophilic material or coating and a hydrophobic or more hydrophobic material or coating may be 5° to 100°, e.g., 5° to 80°, 5° to 60°, 5° to 50°, 5° to 40°, 5° to 30°, 5° to 20°, 10° to 75°, 15° to 70°, 20° to 65°, 25° to 60°, 30 to 50°, 35° to 45°, e.g., 5°, 6°, 7°, 8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60, 65°, 70°, 75°, 80°, 85°, 90°, 95°, or 100°.

[0211] Surfaces may also be coated with various functional materials, e.g., metals or other electrically or magnetically conducting materials. For example, a surface may include a metal coating for electrical connectivity, detection, or resistive heating. Alternatively, such elements may be physically incorporated into a device or placed in physical contact with a device.

[0212] Surface properties may also be modified after application. Such methods include exposure to UV, ozone, plasma e.g., oxygen, argon, etc.), UV photografting and UV induced photo-catalytic oxidation. These and other methods can alter the properties of the surface {e.g., wettability such as hydrophilicity, fluorophilicity, or hydrophobicity) or add an additional layer {e.g., biomolecules) to the surface.

[0213] The above discussion centers on the water contact angle. It will be understood that liquids employed may not be water, or even aqueous. Accordingly, the actual contact angle of a liquid on a surface may differ from the water contact angle. Furthermore, the determination of a water contact angle of a material or coating can be made on that material or coating when not incorporated into a device.

[0214] Methods of Detection

[0215] In some embodiments, the methods described herein include detecting, e.g., tissue, cells, particulate components thereof, or other analytes. A sensor {e.g., optical, electrical, magnetic, impedance, or fluorescent sensor) in the detector may sense a particular-40-FH12653613.3feature (e.g., fluorescence, charge) or characteristic (e.g., diameter or volume) of sample (e.g., a cell or group of cells in a tissue sample).

[0216] Methods of detection include optical detection, e.g., by visual observation, e.g., using an optical bright-field. In some embodiments, analytes thereof are detectable by light absorbance, scatter, emission, and / or transmission. Additionally, or alternatively, optical detection can include fluorescent detection, e.g., by fluorescent microscopy. In still further embodiments, methods of the disclosure include detection of analytes having electrical or magnetic labels or properties. In some embodiments, the device includes a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of detectors. Detectors may or may not be integrated with the device. In some embodiments, the substrate layer and / or fluid interface layer may be transparent, or include transparent portions, e.g., to allow for visualization, imaging, or detection. Substrate layers or fluidic interface layers, or portions thereof, may include transparent materials such as glass, quartz, polystyrene, polyethylene terephthalate, etc. The detection methods described herein may be automated, e.g., including robotic systems.

[0217] A variety of analytes, e.g., tissue, cell, or particulate component or macromolecular constituent thereof, characteristics can be observed and / or quantified. For example, characteristics such as analyte, e.g., cell, or particulate component or macromolecular constituent thereof, size (e.g., diameter) and shape can be readily observed visually and recorded by image or video acquisition software known in the art. In addition, the number of analytes, e.g., cell or particulate component thereof, can similarly be observed visually, by using detectable labels, or by other optical characteristics (e.g., scatter, absorbance, transmission, emission, such as fluorescence, etc.). In some embodiments, methods of the disclosure include observing the presence and / or intensity of a fluorescently or ionically tagged antigen-binding molecule bound to a biological antigen (e.g., a protein or nucleic acid, e.g., associated with an intact cell).

[0218] Preparation of Samples

[0219] A variety of steps can be performed to prepare a biological tissue sample for analysis. In some embodiments, a sample is collected or deposited in the device described herein and prepared using a device described herein. In some embodiments, a prepared sample is placed on a substrate layer described herein. Except where indicated otherwise, the preparative steps described below can generally be combined in any manner to appropriately prepare a particular sample for analysis. In some aspects, any of the preparative or processing -41-FH12653613.3steps described can be performed on a sample using a device described herein, e.g., to deliver reagents via a fluid source. For example, the preparing or processing may include but is not limited to steps for fixing, embedding, staining, crosslinking, permeabilizing the sample, providing and / or removing reagents (e.g., probes, enzymes, buffers, etc.) or any combinations thereof.

[0220] A biological tissue sample can be harvested from a subject (e.g., via surgical biopsy, whole subject sectioning), grown in vitro on a growth substrate or culture dish as a population of cells, or prepared as a tissue slice or tissue section. Grown samples may be sufficiently thin for analysis without further processing steps. Alternatively, grown samples, and samples obtained via biopsy or sectioning, can be prepared as thin tissue sections using a mechanical cutting apparatus such as a vibrating blade microtome. As another alternative, in some embodiments, a thin tissue section can be prepared by applying a touch imprint of a biological sample to a suitable substrate material.

[0221] The thickness of the tissue section can be a fraction of (e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1) the maximum cross-sectional dimension of a cell. However, tissue sections having a thickness that is larger than the maximum cross-section cell dimension can also be used. For example, cryostat sections can be used, which can be, e.g., from about 10 pm to about 20 pm thick.

[0222] More generally, the thickness of a tissue section typically depends on the method used to prepare the section and the physical characteristics of the tissue, and therefore sections having a wide variety of different thicknesses can be prepared and used. For example, the thickness of the tissue section can be at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 1.0, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 20, 30, 40, or 50 pm. Thicker sections can also be used if desired or convenient, e.g., at least 70, 80, 90, or 100 pm or more. Typically, the thickness of a tissue section is about 1-100 pm, 1-50 pm, 1-30 pm, 1-25 pm, 1-20 pm, 1-15 pm, 1-10 pm, 2-8 pm, 3-7 pm, or 4-6 pm, but as mentioned above, sections with thicknesses larger or smaller than these ranges can also be analyzed.

[0223] Multiple sections can also be obtained from a single biological sample. For example, multiple tissue sections can be obtained from a surgical biopsy sample by performing serial sectioning of the biopsy sample using a sectioning blade. Spatial information among the serial sections can be preserved in this manner, and the sections can-42-FH12653613.3be analyzed successively to obtain three-dimensional information about the biological sample.

[0224] In some embodiments, the biological tissue sample (e.g., a tissue section as described above) can be prepared by deep freezing at a temperature suitable to maintain or preserve the integrity (e.g., the physical characteristics) of the tissue structure. Such a temperature can be, e.g., less than -20° C., or less than -25° C., -30° C., -40° C., -50° C., -60° C„ -70° C„ 80° C. -90° C„ -100° C„ -110° C„ -120° C„ -130° C„ -140° C„ -150° C„ -160° C„ -170° C„ -180° C„ -190° C„ or -200° C. The frozen tissue sample can be sectioned, e.g., thinly sliced, onto a substrate surface using any number of suitable methods. For example, a tissue sample can be prepared using a chilled microtome (e.g., a cryostat) set at a temperature suitable to maintain both the structural integrity of the tissue sample and the chemical properties of the nucleic acids in the sample. Such a temperature can be, e.g., less than -15° C., less than -20° C., or less than -25° C. A sample can be snap frozen in isopentane and liquid nitrogen. Frozen samples can be stored in a sealed container prior to embedding.

[0225] Fixation and Postfixation

[0226] In some embodiments, the biological sample can be prepared using formalinfixation and paraffin-embedding (FFPE), which are established methods. In some embodiments, cell suspensions and other non-tissue samples can be prepared using formalinfixation and paraffin-embedding. Following fixation of the sample and embedding in a paraffin or resin block, the sample can be sectioned as described above. Prior to analysis, the paraffin-embedding material can be removed from the tissue section (e.g., deparaffinization) by incubating the tissue section in an appropriate solvent (e.g., xylene) followed by a rinse (e.g., 99.5% ethanol for 2 minutes, 96% ethanol for 2 minutes, and 70% ethanol for 2 minutes).

[0227] As an alternative to formalin fixation described above, a biological sample can be fixed in any of a variety of other fixatives to preserve the biological structure of the sample prior to analysis. For example, a sample can be fixed via immersion in ethanol, methanol, acetone, paraformaldehyde (PFA)-Triton, and combinations thereof.

[0228] In some embodiments, acetone fixation is used with fresh frozen samples, which can include, but are not limited to, cortex tissue, mouse olfactory bulb, human brain tumor, human post-mortem brain, and breast cancer samples. When acetone fixation is -43-FH12653613.3performed, pre-permeabilization steps (described below) may not be performed. Alternatively, acetone fixation can be performed in conjunction with permeabilization steps.

[0229] In some embodiments, the methods provided herein includes one or more post-fixing (also referred to as postfixation) steps. In some embodiments, one or more postfixing step is performed after contacting a sample with a polynucleotide disclosed herein, e.g., one or more probes such as a circular or padlock probe. In some embodiments, one or more post-fixing step is performed after a hybridization complex including a probe and a target is formed in a sample. In some embodiments, one or more post-fixing step is performed prior to a ligation reaction disclosed herein, such as the ligation to circularize a padlock probe.

[0230] In some embodiments, one or more post-fixing step is performed after contacting a sample with a binding or labelling agent (e.g., an antibody or antigen binding fragment thereof) for a non-nucleic acid analyte such as a protein analyte. The labelling agent can include a nucleic acid molecule (e.g., reporter oligonucleotide) including a sequence corresponding to the labelling agent and therefore corresponds to (e.g., uniquely identifies) the analyte. In some embodiments, the labelling agent can include a reporter oligonucleotide including one or more barcode sequences.

[0231] A post-fixing step may be performed using any suitable fixation reagent disclosed herein, for example, 3% (w / v) paraformaldehyde in DEPC-PBS.

[0232] Embedding

[0233] As an alternative to paraffin embedding described above, a biological sample can be embedded in any of a variety of other embedding materials to provide structural substrate to the sample prior to sectioning and other handling steps. In some cases, the embedding material can be removed e.g., prior to analysis of tissue sections obtained from the sample. Suitable embedding materials include, but are not limited to, waxes, resins (e.g., methacrylate resins), epoxies, and agar.

[0234] In some embodiments, the biological sample can be embedded in a matrix (e.g., a hydrogel matrix). Embedding the sample in this manner typically involves contacting the biological sample with a hydrogel such that the biological sample becomes surrounded by the hydrogel. For example, the sample can be embedded by contacting the sample with a suitable polymer material, and activating the polymer material to form a hydrogel. In some-44-FH12653613.3embodiments, the hydrogel is formed such that the hydrogel is internalized within the biological sample.

[0235] In some embodiments, the biological sample is immobilized in the hydrogel via cross-linking of the polymer material that forms the hydrogel. Cross-linking can be performed chemically and / or photochemically, or alternatively by any other hydrogelformation method known in the art.

[0236] The composition and application of the hydrogel-matrix to a biological sample typically depends on the nature and preparation of the biological sample (e.g., sectioned, nonsectioned, type of fixation). As one example, where the biological sample is a tissue section, the hydrogel-matrix can include a monomer solution and an ammonium persulfate (APS) initiator / tetramethylethylenediamine (TEMED) accelerator solution. As another example, where the biological sample consists of cells (e.g., cultured cells or cells disassociated from a tissue sample), the cells can be incubated with the monomer solution and APS / TEMED solutions. For cells, hydrogel-matrix gels are formed in compartments, including but not limited to devices used to culture, maintain, or transport the cells. For example, hydrogelmatrices can be formed with monomer solution plus APS / TEMED added to the compartment to a depth ranging from about 0.1 pm to about 2 mm.

[0237] Additional methods and aspects of hydrogel embedding of biological samples are described for example in Chen et al., Science 347(6221):543-548, 2015, the entire contents of which are incorporated herein by reference.

[0238] Staining and Immunohistochemistry (INC)

[0239] To facilitate visualization, biological samples can be stained using a wide variety of stains and staining techniques. In some embodiments, for example, a sample can be stained using any number of stains and / or immunohistochemical reagents. One or more staining steps may be performed to prepare or process a biological sample for an assay described herein or may be performed during and / or after an assay. In some instances, the provided methods and devices for the reversible assembly and use of a flowcell allow access to the sample after performing fluidic operations. In some cases, the provided flowcell can be sealed then access can be gained to the sample without disrupting the sample (e.g., to perform staining or IHC after performing other fluidic steps of an assay). In some embodiments, the sample can be contacted with one or more nucleic acid stains, membrane stains (e.g., cellular or nuclear membrane), cytological stains, or combinations thereof. In some examples, the -45-FH12653613.3stain may be specific to proteins, phospholipids, DNA (e.g., dsDNA, ssDNA), RNA, an organelle or compartment of the cell. The sample may be contacted with one or more labeled antibodies (e.g., a primary antibody specific for the analyte of interest and a labeled secondary antibody specific for the primary antibody). In some embodiments, cells in the sample can be segmented using one or more images taken of the stained sample.

[0240] In some embodiments, the stain is performed using a lipophilic dye. In some examples, the staining is performed with a lipophilic carbocyanine or aminostyryl dye, or analogs thereof (e.g., Dil, DiO, DiR, DiD). Other cell membrane stains may include FM and RH dyes or immunohistochemical reagents specific for cell membrane proteins. In some examples, the stain may include but is not limited to, acridine orange, acid fuchsin, Bismarck brown, carmine, Coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsine, haematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, ruthenium red, propidium iodide, rhodamine (e.g., rhodamine B), or safranine, or derivatives thereof. In some embodiments, the sample may be stained with haematoxylin and eosin (H&E).

[0241] In some embodiments, biological samples can be destained. Methods of destaining or discoloring a biological sample are known in the art, and generally depend on the nature of the stain(s) applied to the sample. For example, in some embodiments, one or more immunofluorescent stains are applied to the sample via antibody coupling. Such stains can be removed using techniques such as cleavage of disulfide linkages via treatment with a reducing agent and detergent washing, chaotropic salt treatment, treatment with antigen retrieval solution, and treatment with an acidic glycine buffer. Methods for multiplexed staining and destaining are described, for example, in Bolognesi et al., J. Histochem.Cytochem. 2017; 65(8): 431-444, Lin et al., Nat Commun. 2015; 6:8390, Pirici et al., J. Histochem. Cytochem. 2009; 57:567-75, and Glass et al., J. Histochem. Cytochem. 2009; 57:899-905, the entire contents of each of which are incorporated herein by reference.

[0242] Isometric Expansion

[0243] In some embodiments, a biological sample embedded in a matrix (e.g., a hydrogel) can be isometrically expanded. Isometric expansion methods that can be used include hydration, a preparative step in expansion microscopy, as described in Chen et al., Science 347(6221):543-548, 2015.-46-FH12653613.3

[0244] Isometric expansion can be performed by anchoring one or more components of a biological sample (e.g., nucleic acids, proteins) to a gel, followed by gel formation, proteolysis, and swelling. In some embodiments, analytes in the sample, products of the analytes, and / or probes associated with analytes in the sample can be anchored to the matrix (e.g., hydrogel). Isometric expansion of the biological sample can occur prior to immobilization of the biological sample on a substrate, or after the biological sample is immobilized to a substrate. In some embodiments, the isometrically expanded biological sample can be removed from the substrate prior to contacting the substrate with probes disclosed herein.

[0245] Isometric expansion of the sample can increase the spatial resolution of the subsequent analysis of the sample. The increased resolution in spatial profiling can be determined by comparison of an isometrically expanded sample with a sample that has not been isometrically expanded.

[0246] In some embodiments, a biological sample is isometrically expanded to a size at least 2x, 2. lx, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3x, 3. lx, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4x, 4.1x, 4.2x, 4.3x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, or 4.9xits nonexpanded size. In some embodiments, the sample is isometrically expanded to at least 2x and less than 20x of its non-expanded size.

[0247] Crosslinking and De-Crosslinking

[0248] In some embodiments, the biological sample is reversibly cross-linked. In some aspects, the analytes, polynucleotides and / or product of an analyte or a probe bound thereto can be anchored to a polymer matrix. For example, the polymer matrix can be a hydrogel. In some embodiments, portions of the sample can be modified to contain functional groups that can be used as an anchoring site to attach the polynucleotide probes and / or amplification product to a polymer matrix. In some embodiments, a modified probe including oligo dT may be used to bind to mRNA molecules of interest, followed by reversible crosslinking of the mRNA molecules.

[0249] In some embodiments, a hydrogel can include hydrogel subunits, such as, but not limited to, acrylamide, bis-acrylamide, polyacrylamide and derivatives thereof, polyethylene glycol) and derivatives thereof (e.g. PEG-acrylate (PEG-DA), PEG-RGD), gelatin-methacryloyl (GelMA), methacrylated hyaluronic acid (MeHA), poly aliphatic polyurethanes, polyether polyurethanes, polyester polyurethanes, polyethylene copolymers,-47-FH12653613.3polyamides, polyvinyl alcohols, polypropylene glycol, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate), and poly(hydroxyethyl methacrylate), collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin, alginate, protein polymers, methylcellulose, and the like, and combinations thereof.

[0250] In some embodiments, a hydrogel includes a hybrid material, e.g., the hydrogel material includes elements of both synthetic and natural polymers. Examples of suitable hydrogels are described, for example, in U.S. Pat. Nos. 6,391,937, 9,512,422, and 9,889,422, and in U.S. Patent Application Publication Nos. 2017 / 0253918, 2018 / 0052081 and 2010 / 0055733, the entire contents of each of which are incorporated herein by reference.

[0251] In some embodiments, the hydrogel can form the substrate. In some embodiments, the substrate includes a hydrogel and one or more second materials. In some embodiments, the hydrogel is placed on top of one or more second materials. For example, the hydrogel can be pre-formed and then placed on top of, underneath, or in any other configuration with one or more second materials. In some embodiments, hydrogel formation occurs after contacting one or more second materials during formation of the substrate.Hydrogel formation can also occur within a structure (e.g., wells, ridges, projections, and / or markings) located on a substrate.

[0252] In some embodiments, hydrogel formation on a substrate occurs before, contemporaneously with, or after the sample is in the device. For example, hydrogel formation can be performed on the sample on the substrate layer.

[0253] In some embodiments, hydrogel formation occurs within a biological sample. In some embodiments, a biological sample (e.g., tissue section) is embedded in a hydrogel. In some embodiments, hydrogel subunits are infused into the biological sample, and polymerization of the hydrogel is initiated by an external or internal stimulus.

[0254] In embodiments in which a hydrogel is formed within a biological sample, functionalization chemistry can be used. In some embodiments, functionalization chemistry includes hydrogel-tissue chemistry (HTC). Any hydrogel-tissue backbone (e.g., synthetic or native) suitable for HTC can be used for anchoring biological macromolecules and modulating functionalization. Non-limiting examples of methods using HTC backbone variants include CHARITY, PACT, ExM, SWITCH and ePACT. In some embodiments, hydrogel formation within a biological sample is permanent. For example, biological macromolecules can permanently adhere to the hydrogel allowing multiple rounds of -48-FH12653613.3interrogation. In some embodiments, hydrogel formation within a biological sample is reversible.

[0255] In some embodiments, a method disclosed herein includes de-crosslinking the reversibly cross-linked biological sample. The de-crosslinking does not need to be complete. In some embodiments, only a portion of crosslinked molecules in the reversibly cross-linked biological sample are de-crosslinked and allowed to migrate.

[0256] Tissue Permeabilization and Treatment

[0257] In some embodiments, a biological sample can be permeabilized to facilitate transfer of analytes out of the sample, and / or to facilitate transfer of species (such as probes) into the sample. If a sample is not permeabilized sufficiently, the amount of analyte captured from the sample may be too low to enable adequate analysis. Conversely, if the tissue sample is too permeable, the relative spatial relationship of the analytes within the tissue sample can be lost. Hence, a balance between permeabilizing the tissue sample enough to obtain good signal intensity while still maintaining the spatial resolution of the analyte distribution in the sample is desirable.

[0258] In general, a biological sample can be permeabilized by exposing the sample to one or more permeabilizing agents. Suitable agents for this purpose include, but are not limited to, organic solvents (e.g., acetone, ethanol, and methanol), cross-linking agents (e.g., paraformaldehyde), detergents (e.g., saponin, Triton X100™ or Tween-20™), and enzymes (e.g., trypsin, proteases). In some embodiments, the biological sample can be incubated with a cellular permeabilizing agent to facilitate permeabilization of the sample. Additional methods for sample permeabilization are described, for example, in Jamur et al., Method Mol. Biol. 588:63-66, 2010, the entire contents of which are incorporated herein by reference. Any suitable method for sample permeabilization can generally be used in connection with the samples described herein.

[0259] In some embodiments, the biological sample can be permeabilized by adding one or more lysis reagents to the sample. Examples of suitable lysis agents include, but are not limited to, bioactive reagents such as lysis enzymes that are used for lysis of different cell types, e.g., gram positive or negative bacteria, plants, yeast, mammalian, such as lysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase, and a variety of other commercially available lysis enzymes.-49-FH12653613.3

[0260] Other lysis agents can additionally or alternatively be added to the biological sample to facilitate permeabilization. For example, surfactant-based lysis solutions can be used to lyse sample cells. Lysis solutions can include ionic surfactants such as, for example, sarcosyl and sodium dodecyl sulfate (SDS). More generally, chemical lysis agents can include, without limitation, organic solvents, chelating agents, detergents, surfactants, and chaotropic agents.

[0261] In some embodiments, the biological sample can be permeabilized by nonchemical permeabilization methods. Non-chemical permeabilization methods are known in the art. For example, non-chemical permeabilization methods that can be used include, but are not limited to, physical lysis techniques such as electroporation, mechanical permeabilization methods (e.g., bead beating using a homogenizer and grinding balls to mechanically disrupt sample tissue structures), acoustic permeabilization e.g., sonication), and thermal lysis techniques such as heating to induce thermal permeabilization of the sample.

[0262] Additional reagents can be added to a biological sample to perform various functions prior to analysis of the sample. In some embodiments, Dnase and Rnase inactivating agents or inhibitors such as proteinase K, and / or chelating agents such as EDTA, can be added to the sample. For example, a method disclosed herein may include a step for increasing accessibility of a nucleic acid for binding, e.g., a denaturation step to opening up DNA in a cell for hybridization by a probe. For example, proteinase K treatment may be used to free up DNA with proteins bound thereto.

[0263] Analytes

[0264] The methods and compositions disclosed herein can be used to detect and analyze a wide variety of different analytes. In some aspects, an analyte can include any biological substance, structure, moiety, or component to be analyzed. In some aspects, a target disclosed herein may similarly include any analyte of interest. In some examples, a target or analyte can be directly or indirectly detected.

[0265] Analytes can be derived from a specific type of cell and / or a specific sub-cellular region. For example, analytes can be derived from cytosol, from cell nuclei, from mitochondria, from microsomes, and more generally, from any other compartment, organelle, or portion of a cell. Permeabilizing agents that specifically target certain cell compartments and organelles can be used to selectively release analytes from cells for analysis, and / or allow -50-FH12653613.3access of one or more reagents e.g., probes for analyte detection) to the analytes in the cell or cell compartment or organelle.

[0266] The analyte may include any biomolecule or chemical compound, including a macromolecule such as a protein or peptide, a lipid or a nucleic acid molecule, or a small molecule, including organic or inorganic molecules. The analyte may be a cell or a microorganism, including a virus, or a fragment or product thereof. An analyte can be any substance or entity for which a specific binding partner (e.g., an affinity binding partner) can be developed. Such a specific binding partner may be a nucleic acid probe (for a nucleic acid analyte) and may lead directly to the generation of a product. Alternatively, the specific binding partner may be coupled to a nucleic acid, which may be detected.

[0267] Analytes of particular interest may include nucleic acid molecules, such as DNA (e.g., genomic DNA, mitochondrial DNA, plastid DNA, viral DNA, etc.) and RNA (e.g., mRNA, microRNA, rRNA, snRNA, viral RNA, etc.), and synthetic and / or modified nucleic acid molecules, e.g., including nucleic acid domains including or consisting of synthetic or modified nucleotides such as LNA, PNA, morpholino, etc.), proteinaceous molecules such as peptides, polypeptides, proteins or prions or any molecule which includes a protein or polypeptide component, etc., or fragments thereof, or a lipid or carbohydrate molecule, or any molecule which include a lipid or carbohydrate component. The analyte may be a single molecule or a complex that contains two or more molecular subunits, e.g., including but not limited to protein-DNA complexes, which may or may not be covalently bound to one another, and which may be the same or different. Thus, in addition to cells or microorganisms, such a complex analyte may also be a protein complex or protein interaction. Such a complex or interaction may thus be a homo- or hetero-multimer.Aggregates of molecules, e.g., proteins may also be target analytes, for example aggregates of the same protein or different proteins. The analyte may also be a complex between proteins or peptides and nucleic acid molecules such as DNA or RNA, e.g., interactions between proteins and nucleic acids, e.g., regulatory factors, such as transcription factors, and DNA or RNA.

[0268] Endogenous Analytes

[0269] In some embodiments, an analyte herein is endogenous to a biological sample and can include nucleic acid analytes and non-nucleic acid analytes. Methods and compositions disclosed herein can be used to analyze nucleic acid analytes (e.g., using a nucleic acid probe or probe set that directly or indirectly hybridizes to a nucleic acid analyte)-51-FH12653613.3and / or non-nucleic acid analytes (e.g., using a labelling agent that includes a reporter oligonucleotide and binds directly or indirectly to a non-nucleic acid analyte) in any suitable combination.

[0270] Examples of non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, viral coat proteins, extracellular and intracellular proteins, antibodies, and antigen binding fragments.

[0271] Examples of nucleic acid analytes include DNA analytes such as singlestranded DNA (ssDNA), double- stranded DNA (dsDNA), genomic DNA, methylated DNA, specific methylated DNA sequences, fragmented DNA, mitochondrial DNA, in situ synthesized PCR products, and RNA / DNA hybrids. The DNA analyte can be a transcript of another nucleic acid molecule (e.g., DNA or RNA such as mRNA) present in a tissue sample.

[0272] Examples of nucleic acid analytes also include RNA analytes such as various types of coding and non-coding RNA. Examples of the different types of RNA analytes include messenger RNA (mRNA), including a nascent RNA, a pre-mRNA, a primarytranscript RNA, and a processed RNA, such as a capped mRNA (e.g., with a 5' 7-methyl guanosine cap), a polyadenylated mRNA (poly-A tail at the 3' end), and a spliced mRNA in which one or more introns have been removed. Also included in the analytes disclosed herein are non-capped mRNA, a non-polyadenylated mRNA, and a non-spliced mRNA. The RNA analyte can be a transcript of another nucleic acid molecule (e.g., DNA or RNA such as viral RNA) present in a tissue sample. Examples of a non-coding RNAs (ncRNA) that is not translated into a protein include transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), as well as small non-coding RNAs such as microRNA (miRNA), small interfering RNA (siRNA), Piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), extracellular RNA (exRNA), small Cajal body-specific RNAs (scaRNAs), and the long ncRNAs such as Xist and HOTAIR.

[0273] In some embodiments described herein, an analyte may be a denatured nucleic acid, wherein the resulting denatured nucleic acid is single- stranded. The nucleic acid may be denatured, for example, optionally using formamide, heat, or both formamide and heat. In some embodiments, the nucleic acid is not denatured for use in a method disclosed herein.-52-FH12653613.3

[0274] In certain embodiments, an analyte can be extracted from a live cell.Processing conditions can be adjusted to ensure that a biological sample remains live during analysis, and analytes are extracted from (or released from) live cells of the sample. Live cell-derived analytes can be obtained only once from the sample or can be obtained at intervals from a sample that continues to remain in viable condition.

[0275] Methods and compositions disclosed herein can be used to analyze any number of analytes. For example, the number of analytes that are analyzed can be at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 100, at least about 1,000, at least about 10,000, at least about 100,000 or more different analytes present in a region of the sample or within an individual feature of the substrate.

[0276] In any embodiment described herein, the analyte includes a target sequence. In some embodiments, the target sequence may be endogenous to the sample, generated in the sample, added to the sample, or associated with an analyte in the sample. In some embodiments, the target sequence is a single-stranded target sequence (e.g., a sequence in a rolling circle amplification product). In some embodiments, the analytes include one or more single-stranded target sequences. In one aspect, a first single- stranded target sequence is not identical to a second single- stranded target sequence. In another aspect, a first single- stranded target sequence is identical to one or more second single-stranded target sequence. In some embodiments, the one or more second single- stranded target sequence is included in the same analyte (e.g., nucleic acid) as the first single- stranded target sequence. Alternatively, the one or more second single- stranded target sequence is included in a different analyte (e.g., nucleic acid) from the first single- stranded target sequence.

[0277] Labelling Agents

[0278] In some embodiments, provided herein are methods and compositions for analyzing endogenous analytes (e.g., RNA, ssDNA, and cell surface or intracellular proteins and / or metabolites) in a sample using one or more labelling agents. In some embodiments, an analyte labelling agent may include an agent that interacts with an analyte (e.g., an endogenous analyte in a sample). In some embodiments, the labelling agents can include a reporter oligonucleotide that is indicative of the analyte or portion thereof interacting with the -53-FH12653613.3labelling agent. For example, the reporter oligonucleotide may include a barcode sequence that permits identification of the labelling agent. In some cases, the sample contacted by the labelling agent can be further contacted with a probe (e.g., a single- stranded probe sequence), that hybridizes to a reporter oligonucleotide of the labelling agent, in order to identify the analyte associated with the labelling agent. In some embodiments, the analyte labelling agent includes an analyte binding moiety and a labelling agent barcode domain including one or more barcode sequences, e.g., a barcode sequence that corresponds to the analyte binding moiety and / or the analyte. An analyte binding moiety barcode includes a barcode that is associated with or otherwise identifies the analyte binding moiety. In some embodiments, by identifying an analyte binding moiety by identifying its associated analyte binding moiety barcode, the analyte to which the analyte binding moiety binds can also be identified. An analyte binding moiety barcode can be a nucleic acid sequence of a given length and / or sequence that is associated with the analyte binding moiety. An analyte binding moiety barcode can generally include any of the variety of aspects of barcodes described herein.

[0279] In some embodiments, the method includes one or more post-fixing (also referred to as post-fixation) steps after contacting the sample with one or more labelling agents.

[0280] In the methods and devices described herein, one or more labelling agents capable of binding to or otherwise coupling to one or more features may be used to characterize analytes, cells and / or cell features. In some instances, cell features include cell surface features. Analytes may include, but are not limited to, a protein, a receptor, an antigen, a surface protein, a transmembrane protein, a cluster of differentiation protein, a protein channel, a protein pump, a carrier protein, a phospholipid, a glycoprotein, a glycolipid, a cell-cell interaction protein complex, an antigen-presenting complex, a major histocompatibility complex, an engineered T-cell receptor, a T-cell receptor, a B-cell receptor, a chimeric antigen receptor, a gap junction, an adherens junction, or any combination thereof. In some instances, cell features may include intracellular analytes, such as proteins, protein modifications (e.g., phosphorylation status or other post-translational modifications), nuclear proteins, nuclear membrane proteins, or any combination thereof.

[0281] In some embodiments, an analyte binding moiety may include any molecule or moiety capable of binding to an analyte (e.g., a biological analyte, e.g., a macromolecular constituent). A labelling agent may include, but is not limited to, a protein, a peptide, an -54-FH12653613.3antibody (or an epitope binding fragment thereof), a lipophilic moiety (such as cholesterol), a cell surface receptor binding molecule, a receptor ligand, a small molecule, a bi- specific antibody, a bi-specific T-cell engager, a T-cell receptor engager, a B-cell receptor engager, a pro-body, an aptamer, a monobody, an affimer, a darpin, and a protein scaffold, or any combination thereof. The labelling agents can include (e.g., are attached to) a reporter oligonucleotide that is indicative of the cell surface feature to which the binding group binds. For example, the reporter oligonucleotide may include a barcode sequence that permits identification of the labelling agent. For example, a labelling agent that is specific to one type of cell feature (e.g., a first cell surface feature) may have coupled thereto a first reporter oligonucleotide, while a labelling agent that is specific to a different cell feature (e.g., a second cell surface feature) may have a different reporter oligonucleotide coupled thereto. For a description of exemplary labelling agents, reporter oligonucleotides, and methods of use, see, e.g., U.S. Pat. No. 10,550,429; U.S. Pat. Pub. 20190177800; and U.S. Pat. Pub. 20190367969, which are each incorporated by reference herein in their entirety.

[0282] In some embodiments, an analyte binding moiety includes one or more antibodies or antigen binding fragments thereof. The antibodies or antigen binding fragments including the analyte binding moiety can specifically bind to a target analyte. In some embodiments, the analyte is a protein (e.g., a protein on a surface of the biological sample (e.g., a cell) or an intracellular protein). In some embodiments, a plurality of analyte labelling agents including a plurality of analyte binding moieties bind a plurality of analytes present in a biological sample. In some embodiments, the plurality of analytes includes a single species of analyte (e.g., a single species of polypeptide). In some embodiments in which the plurality of analytes includes a single species of analyte, the analyte binding moieties of the plurality of analyte labelling agents are the same. In some embodiments in which the plurality of analytes includes a single species of analyte, the analyte binding moieties of the plurality of analyte labelling agents are the different (e.g., members of the plurality of analyte labelling agents can have two or more species of analyte binding moieties, wherein each of the two or more species of analyte binding moieties binds a single species of analyte, e.g., at different binding sites). In some embodiments, the plurality of analytes includes multiple different species of analyte (e.g., multiple different species of polypeptides).

[0283] In other instances, e.g., to facilitate sample multiplexing, a labelling agent that is specific to a particular cell feature may have a first plurality of the labelling agent (e.g., an -55-FH12653613.3antibody or lipophilic moiety) coupled to a first reporter oligonucleotide and a second plurality of the labelling agent coupled to a second reporter oligonucleotide.

[0284] In some aspects, these reporter oligonucleotides may include nucleic acid barcode sequences that permit identification of the labelling agent which the reporter oligonucleotide is coupled to. The selection of oligonucleotides as the reporter may provide advantages of being able to generate significant diversity in terms of sequence, while also being readily attachable to most biomolecules, e.g., antibodies, etc., as well as being readily detected, e.g., using sequencing or array technologies.

[0285] Attachment (coupling) of the reporter oligonucleotides to the labelling agents may be achieved through any of a variety of direct or indirect, covalent or non-covalent associations or attachments. For example, oligonucleotides may be covalently attached to a portion of a labelling agent (such a protein, e.g., an antibody or antibody fragment) using chemical conjugation techniques (e.g., Lightning-Link® antibody labelling kits available from Innova Biosciences), as well as other non-covalent attachment mechanisms, e.g., using biotinylated antibodies and oligonucleotides (or beads that include one or more biotinylated linker, coupled to oligonucleotides) with an avidin or streptavidin linker. Antibody and oligonucleotide biotinylation techniques are available. See, e.g., Fang, et al., “Fluoride-Cleavable Biotinylation Phosphoramidite for 5'-end-Labelling and Affinity Purification of Synthetic Oligonucleotides,” Nucleic Acids Res. Jan. 15, 2003; 31(2):708-715, which is entirely incorporated herein by reference for all purposes. Likewise, protein and peptide biotinylation techniques have been developed and are readily available. See, e.g., U.S. Pat. No. 6,265,552, which is entirely incorporated herein by reference for all purposes.Furthermore, click reaction chemistry may be used to couple reporter oligonucleotides to labelling agents. Commercially available kits, such as those from Thunderlink and Abeam, and techniques common in the art may be used to couple reporter oligonucleotides to labelling agents as appropriate. In another example, a labelling agent is indirectly (e.g., via hybridization) coupled to a reporter oligonucleotide including a barcode sequence that identifies the label agent. For instance, the labelling agent may be directly coupled (e.g., covalently bound) to a hybridization oligonucleotide that includes a sequence that hybridizes with a sequence of the reporter oligonucleotide. Hybridization of the hybridization oligonucleotide to the reporter oligonucleotide couples the labelling agent to the reporter oligonucleotide. In some embodiments, the reporter oligonucleotides are releasable from the -56-FH12653613.3labelling agent, such as upon application of a stimulus. For example, the reporter oligonucleotide may be attached to the labeling agent through a labile bond (e.g., chemically labile, photolabile, thermally labile, etc.) as generally described for releasing molecules from supports elsewhere herein. In some instances, the reporter oligonucleotides described herein may include one or more functional sequences that can be used in subsequent processing, such as an adapter sequence, a unique molecular identifier (UMI) sequence, a sequencer specific flow cell attachment sequence (such as an P5, P7, or partial P5 or P7 sequence), a primer or primer binding sequence, a sequencing primer or primer binding sequence (such as an Rl, R2, or partial R1 or R2 sequence).

[0286] In some cases, the labelling agent can include a reporter oligonucleotide and a label. A label can be fluorophore, a radioisotope, a molecule capable of a colorimetric reaction, a magnetic particle, or any other suitable molecule or compound capable of detection. The label can be conjugated to a labelling agent (or reporter oligonucleotide) either directly or indirectly (e.g., the label can be conjugated to a molecule that can bind to the labelling agent or reporter oligonucleotide). In some cases, a label is conjugated to a first oligonucleotide that is complementary (e.g., hybridizes) to a sequence of the reporter oligonucleotide.

[0287] In some embodiments, multiple different species of analytes (e.g., polypeptides) from the biological sample can be subsequently associated with the one or more physical properties of the biological sample. For example, the multiple different species of analytes can be associated with locations of the analytes in the biological sample. Such information (e.g., proteomic information when the analyte binding moiety (ies) recognizes a polypeptide(s)) can be used in association with other spatial information (e.g., genetic information from the biological sample, such as DNA sequence information, transcriptome information (i.e.., sequences of transcripts), or both). For example, a cell surface protein of a cell can be associated with one or more physical properties of the cell (e.g., a shape, size, activity, or a type of the cell). The one or more physical properties can be characterized by imaging the cell. The cell can be bound by an analyte labelling agent including an analyte binding moiety that binds to the cell surface protein and an analyte binding moiety barcode that identifies that analyte binding moiety. Results of protein analysis in a sample (e.g., a tissue sample or a cell) can be associated with DNA and / or RNA analysis in the sample.

[0288] Products of Endogenous Analyte and / or Labelling Agent-57-FH12653613.3

[0289] In some embodiments, provided herein are methods and compositions for analyzing one or more products of an endogenous analyte and / or a labelling agent in a biological sample. In some embodiments, an endogenous analyte (e.g., a viral or cellular DNA or RNA) or a product (e.g., a hybridization product, a ligation product, an extension product (e.g., by a DNA or RNA polymerase), a replication product, a transcription / reverse transcription product, and / or an amplification product thereof is analyzed. In some aspects, the generation and / or processing of the analytes may be performed in the device and / or the analysis of the analytes may be performed in the device, such as by delivering reagents to a sample via a fluid source. For example, the generation, processing, and analysis may include but is not limited to reactions including hybridizations, ligations, binding, extension, amplifications, or other enzymatic reactions. In some embodiments, a labelling agent that directly or indirectly binds to an analyte in the biological sample is analyzed. In some embodiments, a product (e.g., a hybridization product, a ligation product, an extension product (e.g., by a DNA or RNA polymerase), a replication product, a transcription / reverse transcription product, and / or an amplification product of a labelling agent that directly or indirectly binds to an analyte in the biological sample is analyzed. In some embodiments, the reactions for generating any of the products (e.g., ligation, amplification, extension, hybridization) provided herein are performed in the devices provided herein.

[0290] Hybridization

[0291] In some embodiments, a product of an endogenous analyte and / or a labelling agent is a hybridization product including the pairing of substantially complementary or complementary nucleic acid sequences within two different molecules, one of which is the endogenous analyte or the labelling agent (e.g., reporter oligonucleotide attached thereto). The other molecule can be another endogenous molecule or another labelling agent such as a probe. Pairing can be achieved by any process in which a nucleic acid sequence joins with a substantially or fully complementary sequence through base pairing to form a hybridization complex. For purposes of hybridization, two nucleic acid sequences are “substantially complementary” if at least 60% (e.g., at least 70%, at least 80%, or at least 90%) of their individual bases are complementary to one another.

[0292]

[0290]

[0293] Various probes and probe sets can be hybridized to an endogenous analyte and / or a labelling agent and each probe may include one or more barcode sequences.-58-FH12653613.3Exemplary barcoded probes or probe sets may be based on a padlock probe, a gapped padlock probe, a SNAIL (Splint Nucleotide Assisted Intramolecular Ligation) probe set, a PLAYR (Proximity Ligation Assay for RNA) probe set, a PLISH (Proximity Ligation in situ Hybridization) probe set, and RNA-templated ligation probes. The specific probe or probe set design can vary.

[0294] Ligation

[0295] In some embodiments, a product of an endogenous analyte and / or a labelling agent is a ligation product. In some embodiments, the ligation product is formed between two or more endogenous analytes. In some embodiments, the ligation product is formed between an endogenous analyte and a labelling agent. In some embodiments, the ligation product is formed between two or more labelling agent. In some embodiments, the ligation product is an intramolecular ligation of an endogenous analyte. In some embodiments, the ligation product is an intramolecular ligation of a labelling agent, for example, the circularization of a circularizable probe or probe set upon hybridization to a target sequence. The target sequence can be included in an endogenous analyte (e.g., nucleic acid such as a genomic DNA or mRNA) or a product thereof (e.g., cDNA from a cellular mRNA transcript), or in a labelling agent (e.g., the reporter oligonucleotide) or a product thereof.

[0296] In some embodiments, the ligation involves chemical ligation. In some embodiments, the ligation involves template dependent ligation. In some embodiments, the ligation involves template independent ligation. In some embodiments, the ligation involves enzymatic ligation.

[0297] In some embodiments, the enzymatic ligation involves use of a ligase. In some aspects, the ligase used herein includes an enzyme that is commonly used to join polynucleotides together or to join the ends of a single polynucleotide. An RNA ligase, a DNA ligase, or another variety of ligase can be used to ligate two nucleotide sequences together. Ligases include ATP-dependent double-strand polynucleotide ligases, NAD-i-dependent double-strand DNA or RNA ligases and single-strand polynucleotide ligases, for example any of the ligases described in EC 6.5.1.1 (ATP-dependent ligases), EC 6.5.1.2 (NAD+-dependent ligases), EC 6.5.1.3 (RNA ligases). Specific examples of ligases include bacterial ligases such as E. coli DNA ligase, Tth DNA ligase, Thermococcus sp. (strain 9° N) DNA ligase (9° N™ DNA ligase, New England Biolabs), Taq DNA ligase, Ampligase™ (Epicentre Biotechnologies) and phage ligases such as T3 DNA ligase, T4 DNA ligase and -59-FH12653613.3T7 DNA ligase and mutants thereof. In some embodiments, the ligase is a T4 RNA ligase. In some embodiments, the ligase is a splintR ligase. In some embodiments, the ligase is a single stranded DNA ligase. In some embodiments, the ligase is a T4 DNA ligase. In some embodiments, the ligase is a ligase that has an DNA-splinted DNA ligase activity. In some embodiments, the ligase is a ligase that has an RNA-splinted DNA ligase activity.

[0298] In some embodiments, the ligation herein is a direct ligation. In some embodiments, the ligation herein is an indirect ligation. “Direct ligation” means that the ends of the polynucleotides hybridize immediately adjacently to one another to form a substrate for a ligase enzyme resulting in their ligation to each other (intramolecular ligation).Alternatively, “indirect” means that the ends of the polynucleotides hybridize non- adjacently to one another, i.e.., separated by one or more intervening nucleotides or “gaps”. In some embodiments, said ends are not ligated directly to each other, but instead occurs either via the intermediacy of one or more intervening (so-called “gap” or “gap-filling” (oligo)nucleotides) or by the extension of the 3' end of a probe to “fill” the “gap” corresponding to said intervening nucleotides (intermolecular ligation). In some cases, the gap of one or more nucleotides between the hybridized ends of the polynucleotides may be “filled” by one or more “gap” (oligo)nucleotide(s) which are complementary to a splint, padlock probe, or target nucleic acid. The gap may be a gap of 1 to 60 nucleotides or a gap of 1 to 40 nucleotides or a gap of 3 to 40 nucleotides. In specific embodiments, the gap may be a gap of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides, of any integer (or range of integers) of nucleotides in between the indicated values. In some embodiments, the gap between said terminal regions may be filled by a gap oligonucleotide or by extending the 3' end of a polynucleotide. In some cases, ligation involves ligating the ends of the probe to at least one gap (oligo)nucleotide, such that the gap (oligo)nucleotide becomes incorporated into the resulting polynucleotide. In some embodiments, the ligation herein is preceded by gap filling. In other embodiments, the ligation herein does not require gap filling.

[0299] In some embodiments, ligation of the polynucleotides produces polynucleotides with melting temperature higher than that of unligated polynucleotides. Thus, in some aspects, ligation stabilizes the hybridization complex containing the ligated polynucleotides prior to subsequent steps, including amplification and detection.

[0300] In some aspects, a high fidelity ligase, such as a thermostable DNA ligase (e.g., a Taq DNA ligase), is used. Thermostable DNA ligases are active at elevated-60-FH12653613.3temperatures, allowing further discrimination by incubating the ligation at a temperature near the melting temperature (Tm) of the DNA strands. This selectively reduces the concentration of annealed mismatched substrates (expected to have a slightly lower Tm around the mismatch) over annealed fully base-paired substrates. Thus, high-fidelity ligation can be achieved through a combination of the intrinsic selectivity of the ligase active site and balanced conditions to reduce the incidence of annealed mismatched dsDNA.

[0301] In some embodiments, the ligation herein is a proximity ligation of ligating two (or more) nucleic acid sequences that are in proximity with each other, e.g., through enzymatic means (e.g., a ligase). In some embodiments, proximity ligation can include a “gap-filling” step that involves incorporation of one or more nucleic acids by a polymerase, based on the nucleic acid sequence of a template nucleic acid molecule, spanning a distance between the two nucleic acid molecules of interest (see, e.g., U.S. Pat. No. 7,264,929, the entire contents of which are incorporated herein by reference). A wide variety of different methods can be used for proximity ligating nucleic acid molecules, including (but not limited to) “sticky-end” and “blunt-end” ligations. Additionally, single-stranded ligation can be used to perform proximity ligation on a single- stranded nucleic acid molecule. Sticky-end proximity ligations involve the hybridization of complementary single- stranded sequences between the two nucleic acid molecules to be joined, prior to the ligation event itself. Blunt-end proximity ligations generally do not include hybridization of complementary regions from each nucleic acid molecule because both nucleic acid molecules lack a single-stranded overhang at the site of ligation.

[0302] Primer Extension and Amplification

[0303] In some embodiments, a product is a primer extension product of an analyte, a labelling agent, a probe or probe set bound to the analyte (e.g., a circularizable probe bound to genomic DNA, mRNA, or cDNA), or a probe or probe set bound to the labelling agent (e.g., a circularizable probe bound to one or more reporter oligonucleotides from the same or different labelling agents).

[0304] A primer is generally a single- stranded nucleic acid sequence having a 3' end that can be used as a substrate for a nucleic acid polymerase in a nucleic acid extension reaction. RNA primers are formed of RNA nucleotides, and are used in RNA synthesis, while DNA primers are formed of DNA nucleotides and used in DNA synthesis. Primers can also include both RNA nucleotides and DNA nucleotides (e.g., in a random or designed pattern).-61-FH12653613.3Primers can also include other natural or synthetic nucleotides described herein that can have additional functionality. In some examples, DNA primers can be used to prime RNA synthesis and vice versa (e.g., RNA primers can be used to prime DNA synthesis). Primers can vary in length. For example, primers can be about 6 bases to about 120 bases. For example, primers can include up to about 25 bases. A primer, may in some cases, refer to a primer binding sequence. A primer extension reaction generally refers to any method where two nucleic acid sequences become linked (e.g., hybridized) by an overlap of their respective terminal complementary nucleic acid sequences (z.e.., for example, 3' termini). Such linking can be followed by nucleic acid extension (e.g., an enzymatic extension) of one, or both termini using the other nucleic acid sequence as a template for extension. Enzymatic extension can be performed by an enzyme including, but not limited to, a polymerase and / or a reverse transcriptase.

[0305] In some embodiments, a product of an endogenous analyte and / or a labelling agent is an amplification product of one or more polynucleotides, for instance, a circular probe or circularizable probe or probe set. In some embodiments, the amplifying is achieved by performing rolling circle amplification (RCA). In other embodiments, a primer that hybridizes to the circular probe or circularized probe is added and used as such for amplification. In some embodiments, the RCA includes a linear RCA, a branched RCA, a dendritic RCA, or any combination thereof.

[0306] In some embodiments, the amplification is performed at a temperature between or between about 20° C. and about 60° C. In some embodiments, the amplification is performed at a temperature between or between about 30° C. and about 40° C. In some aspects, the amplification step, such as the rolling circle amplification (RCA) is performed at a temperature between at or about 25° C. and at or about 50° C., such as at or about 25° C., 27° C„ 29° C„ 31° C„ 33° C„ 35° C„ 37° C„ 39° C„ 41° C„ 43° C„ 45° C„ 47° C„ or 49° C.

[0307] In some embodiments, upon addition of a DNA polymerase in the presence of appropriate dNTP precursors and other cofactors, a primer is elongated to produce multiple copies of the circular template. This amplification step can utilize isothermal amplification or non-isothermal amplification.

[0308] Target Sequences-62-FH12653613.3

[0309] A target sequence for a probe disclosed herein may be included in any analyte disclose herein, including an endogenous analyte (e.g., a viral or cellular nucleic acid), a labelling agent, or a product of an endogenous analyte and / or a labelling agent.

[0310] In some aspects, one or more of the target sequences includes one or more barcode(s), e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more barcodes. Barcodes can spatially-resolve molecular components found in biological samples, for example, within a cell or a tissue sample. A barcode can be attached to an analyte or to another moiety or structure in a reversible or irreversible manner. A barcode can be added to, for example, a fragment of a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before or during sequencing of the sample. Barcodes can allow for identification and / or quantification of individual sequencing-reads (e.g., a barcode can be or can include a unique molecular identifier or “UMI”). In some aspects, a barcode includes about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 nucleotides.

[0311] In some embodiments, a barcode includes two or more sub-barcodes that together function as a single barcode. For example, a polynucleotide barcode can include two or more polynucleotide sequences (e.g., sub-barcodes) that are separated by one or more nonbarcode sequences. In some embodiments, the one or more barcode(s) can also provide a platform for targeting functionalities, such as oligonucleotides, oligonucleotide-antibody conjugates, oligonucleotide-streptavidin conjugates, modified oligonucleotides, affinity purification, detectable moieties, enzymes, enzymes for detection assays or other functionalities, and / or for detection and identification of the polynucleotide.

[0312] In some embodiments, in a barcode sequencing method, barcode sequences are detected for identification of other molecules including nucleic acid molecules (DNA or RNA) longer than the barcode sequences themselves, as opposed to direct sequencing of the longer nucleic acid molecules. In some embodiments, a N-mer barcode sequence includes 4N complexity given a sequencing read of N bases, and a much shorter sequencing read may be required for molecular identification compared to non-barcode sequencing methods such as direct sequencing. For example, 1024 molecular species may be identified using a 5-nucleotide barcode sequence (45=1024), whereas 8 nucleotide barcodes can be used to identify up to 65,536 molecular species, a number greater than the total number of distinct genes in the human genome. In some embodiments, the barcode sequences contained in the -63-FH12653613.3probes or RCPs are detected, rather than endogenous sequences, which can be an efficient read-out in terms of information per cycle of sequencing. Because the barcode sequences are pre-determined, they can also be designed to feature error detection and correction mechanisms, see, e.g., U.S. Pat. Pub. 20190055594 and WO2019199579A1, which are hereby incorporated by reference in their entirety.

[0313] Assays

[0314] The methods described herein may be useful for analysis methods in which specific reagents are added to a sample. In some embodiments, reagents are added to the sample in the device which include but are not limited to oligonucleotides (e.g., probes, dNTPs, primers), enzymes (e.g., endonucleases to fragment DNA, DNA polymerase enzymes, RNA polymerase, transposase, ligase, proteinase K, reverse transcriptase enzymes, including enzymes with terminal transferase activity, and DNAse), buffers and washes. In some embodiments, optical labels or dyes are added to the sample. In some embodiments, a sample can be collected from the device after performing steps of the assay described herein. In some embodiments, the device is used to perform or prepare sample for in situ analysis methods which include, e.g., in situ hybridization and in situ sequencing. In situ hybridization is a hybridization process in which labeled nucleic acids that are complementary to a specific nucleic acid (e.g., DNA or RNA) sequence in a biological sample hybridize to a portion or section of the sample (e.g., tissue) in which the nucleic acid is present. The methods described herein may be useful for array-based methods in which specific reagents are contacted with a sample. In some embodiments, the surface of the fluidic interface layer or substrate layer may have an array of bound reagents. In some embodiments, a device is used to deliver reagents to the sample which is deposited on the array.

[0315] The labeled nucleic acids, also referred to as probes, are generally short oligonucleotides in which at least a portion of the oligonucleotide is a reverse complement to a target nucleic acid of interest. The probes may include additional components in addition to the hybridization portion. For example, the probes may include additional sequences (e.g., barcode sequences), that are unique labels or identifiers to convey information about the nucleic acid being detected. The probes may further include a label attached thereto, directly or indirectly. The label may be, e.g., an optical label, a molecular label (e.g., an antigen), a radiolabel, or a field attractable label (e.g., electric or magnetic). In some embodiments the-64-FH12653613.3optical label is a fluorescent label, e.g., as used in fluorescence in situ hybridization (FISH). A fluorescent label can be detected by routine optical detection methods known in the art.

[0316] Optical detection may be performed by any detector capable of measuring light (e.g., the emitted, scattered, or attenuated light) from the label. Suitable detectors include, but are not limited to, a spectrometer, a light meter, a photometer, a photodiode, a photomultiplier tube, a CCD array, a CMOS sensor, or a photovoltaic device.

[0317] In situ methods may first include fixing and / or permeabilizing a biological sample (e.g., tissue). The biological sample may be provided in the device, e.g., on a substrate layer. The sample may be permeabilized by adding a fluid, such as a solvent (e.g., acetone and methanol) or a detergent (e.g., TRITON X-100, NP-40, TWEEN 20, saponin, digitonin, and Leucoperm), to the sample. Permeabilization may allow or enhance access of the probes for the intracellular space of the sample.

[0318] In some embodiments, a plurality of probes is used, e.g., for ease of detection and / or signal amplification, such as any probes described herein. For example, a first probe may include a nucleic acid sequence that hybridizes to a target nucleic acid in the sample. A secondary probe that includes a label (e.g., optical label, e.g., fluorescent label) may then be added that hybridizes to the first probe. In some embodiments, a plurality of secondary or higher order (e.g., tertiary, quaternary) detection probes are added. Each probe may be provided by a separate fluid source. Each probe may be provided by a single fluid source that includes a plurality of distinct probes.

[0319] When a probe that includes a detection label is added, the unbound probes with detection labels can be washed away and the signal can be detected, e.g., via fluorescence microscopy.

[0320] In some embodiments, the signal or template target nucleic acid is amplified. In some embodiments, an analyte (e.g., target nucleic acid) can be amplified using an enzyme, e.g., by polymerase chain reaction (PCR) or rolling circle amplification (RCA). The target nucleic acid may be replicated, e.g., by using the probe as a primer to initiate DNA or RNA synthesis. In such an embodiment, one or more fluids are added (e.g., sequentially) to the sample to provide the reagents for nucleic acid synthesis. Suitable reagents include, but are not limited to, probes, primers, nucleotide triphosphates (NTPs, e.g., dNTPs), sequencing terminators, dyes, polymerases, ligases, transcriptases (e.g., reverse transcriptases), labels, and the like.-65-FH12653613.3

[0321] In some embodiments, the methods described herein includes in situ sequencing or sequence detection. One such process includes temporal multiplexing of barcoded probes. In some embodiments, a primary probe or set of primary probes (e.g., 24 primary probes) hybridize to a target nucleic acid (e.g., mRNA) in the sample. Each probe may contain a barcode attached thereto. The barcodes may then be detected by contacting with one or more probes each labeled with a fluorescent label which emits a signal. Each round of barcoding may be initiated by flowing the desired probe from a new fluid source. The labels may be detected using different excitation wavelengths (e.g., 640 nm, 561 nm, or 488 nm) during different barcoding rounds. By compiling the spatiotemporal patterns of each fluorescent signal at a location, the unique set of ordered barcode sequences that corresponds to a particular gene can be determined. Such a method may allow multiplex sequencing of a large number of (e.g., of 100, 1,000, 10,000, or more) nucleic acids, e.g., up to 90,000 transcripts per cell. This method also allows for efficient quantification of low-copy number nucleic acids.

[0322] In some embodiments, the in situ detection and / or in situ sequencing is performed in three dimensions. In this embodiment, the biological sample may be sequence by using a probe that includes a unique gene identifier. The probe may be ligated, thereby allowing extension and amplification of the target sequence. In some embodiments, the amplification product can then be modified with a chemical moiety that polymerizes in the presence of a polymerization initiator. In some embodiments, an amplified product may be embedded within a polymerized matrix (e.g., a hydrogel), thereby creating spatially fixed three-dimensional target analytes of the biological sample.

[0323] In some embodiments, the in situ sequencing includes sequencing by ligation. In this embodiment, fluorescently labeled probes with two known bases followed by degenerate or universal bases hybridize to a temple. A ligase immobilizes the complex and the biological sample is imaged to detect the label on the probe. Following detection, the fluorophore is cleaved from the probe along with several bases, revealing a free 5' phosphate. This process of hybridization, ligation, imaging, and cleavage can be repeating in multiple rounds, thereby allowing identification of, e.g., 2 out of every 5 bases. After a round of probe extension, all probes and anchors are removed and the cycle can begin again with an offset anchor, thus allowing sequencing of a new register of the target.-66-FH12653613.3

[0324] In another embodiment, sequencing by ligation includes labeled probes with a known base (e.g., A, C, T, or G) flanked on each side of the known base by degenerate or universal bases that hybridize to a template (e.g., three or four bases on each side). Each probe contains a different fluorescent label corresponding to each individual base. Each round of sequencing includes hybridizing a probe with a known base, ligation of the probe, detection, and optionally, cleavage of the fluorescent label. Sequencing can be performed in a plus or minus direction, and rounds of sequencing can begin again with an offset anchor, thus allowing sequencing of a new register of the target.

[0325] In some embodiments, detection of one or more analytes (e.g., protein analytes) can be performed using one or more analyte capture agents. In some embodiment, the devices described herein may include one or more analyte capture agents, e.g., an array of oligonucleotides. In some aspects, the array may include a bead array. As used herein, an “analyte capture agent” refers to an agent that interacts with an analyte (e.g., an analyte in a biological sample) and with a capture probe (e.g., a capture probe attached to a substrate or a feature) to identify the analyte. In some embodiments, the analyte capture agent includes: (i) an analyte binding moiety (e.g., that binds to an analyte), for example, an antibody or antigen-binding fragment thereof; (ii) analyte binding moiety barcode; and (iii) an analyte capture sequence. As used herein, the term “analyte binding moiety barcode” refers to a barcode that is associated with or otherwise identifies the analyte binding moiety. As used herein, the term “analyte capture sequence” refers to a region or moiety configured to hybridize to, bind to, couple to, or otherwise interact with a capture domain of a capture probe. In some cases, an analyte binding moiety barcode (or portion thereof) may be able to be removed (e.g., cleaved) from the analyte capture agent. Additional description of analyte capture agents can be found in Section (II)(b)(ix) of WO 2020 / 176788 and / or Section (II)(b)(viii) U.S. Patent Application Publication No. 2020 / 0277663.

[0326] There are at least two methods to associate a spatial barcode with one or more neighboring cells, such that the spatial barcode identifies the one or more cells, and / or contents of the one or more cells, as associated with a particular spatial location. One method is to promote analytes or analyte proxies (e.g., intermediate agents) out of a cell and towards a spatially-barcoded array (e.g., including spatially-barcoded capture probes). Another method is to cleave spatially-barcoded capture probes from an array and promote the spatially-barcoded capture probes towards and / or into or onto the biological sample.-67-FH12653613.3

[0327] In some cases, capture probes may be configured to prime, replicate, and consequently yield optionally barcoded extension products from a template (e.g., a DNA or RNA template, such as an analyte or an intermediate agent (e.g., a ligation product or an analyte capture agent), or a portion thereof), or derivatives thereof (see, e.g., Section (II)(b)(vii) of WO 2020 / 176788 and / or U.S. Patent Application Publication No.2020 / 0277663 regarding extended capture probes). In some cases, capture probes may be configured to form ligation products with a template (e.g., a DNA or RNA template, such as an analyte or an intermediate agent, or portion thereof), thereby creating ligations products that serve as proxies for a template.

[0328] As used herein, an “extended capture probe” refers to a capture probe having additional nucleotides added to the terminus (e.g., 3' or 5' end) of the capture probe thereby extending the overall length of the capture probe. For example, an “extended 3' end” indicates additional nucleotides were added to the most 3' nucleotide of the capture probe to extend the length of the capture probe, for example, by polymerization reactions used to extend nucleic acid molecules including templated polymerization catalyzed by a polymerase (e.g., a DNA polymerase or a reverse transcriptase). In some embodiments, extending the capture probe includes adding to a 3' end of a capture probe a nucleic acid sequence that is complementary to a nucleic acid sequence of an analyte or intermediate agent specifically bound to the capture domain of the capture probe. In some embodiments, the capture probe is extended using reverse transcription. In some embodiments, the capture probe is extended using one or more DNA polymerases. The extended capture probes include the sequence of the capture probe and the sequence of the spatial barcode of the capture probe.

[0329] In some embodiments, extended capture probes are amplified (e.g., in bulk solution or on the array) to yield quantities that are sufficient for downstream analysis, e.g., via DNA sequencing. In some embodiments, extended capture probes (e.g., DNA molecules) act as templates for an amplification reaction (e.g., a polymerase chain reaction).

[0330] Additional variants of spatial analysis methods, including in some embodiments, an imaging step, are described in Section (II)(a) of WO 2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. Analysis of captured analytes (and / or intermediate agents or portions thereof), for example, including sample removal, extension of capture probes, sequencing (e.g., of a cleaved extended capture probe and / or a cDNA molecule complementary to an extended capture probe), sequencing on the array (e.g., using,-68-FH12653613.3for example, in situ hybridization or in situ ligation approaches), temporal analysis, and / or proximity capture, is described in Section (II)(g) of WO 2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. Some quality control measures are described in Section (II)(h) of WO 2020 / 176788 and / or U.S. Patent Application Publication No.2020 / 0277663.

[0331] Spatial information can provide information of biological and / or medical importance. For example, the methods and compositions described herein can allow for: identification of one or more biomarkers (e.g., diagnostic, prognostic, and / or for determination of efficacy of a treatment) of a disease or disorder; identification of a candidate drug target for treatment of a disease or disorder; identification (e.g., diagnosis) of a subject as having a disease or disorder; identification of stage and / or prognosis of a disease or disorder in a subject; identification of a subject as having an increased likelihood of developing a disease or disorder; monitoring of progression of a disease or disorder in a subject; determination of efficacy of a treatment of a disease or disorder in a subject; identification of a patient subpopulation for which a treatment is effective for a disease or disorder; modification of a treatment of a subject with a disease or disorder; selection of a subject for participation in a clinical trial; and / or selection of a treatment for a subject with a disease or disorder.

[0332] Spatial information can provide information of biological importance. For example, the methods and compositions described herein can allow for: identification of transcriptome and / or proteome expression profiles (e.g., in healthy and / or diseased tissue); identification of multiple analyte types in close proximity (e.g., nearest neighbor analysis); determination of up- and / or down-regulated genes and / or proteins in diseased tissue; characterization of tumor microenvironments; characterization of tumor immune responses; characterization of cells types and their co-localization in tissue; and identification of genetic variants within tissues (e.g., based on gene and / or protein expression profiles associated with specific disease or disorder biomarkers).

[0333] Typically, for spatial array-based methods, a substrate layer (e.g., as described herein) functions as a support for direct or indirect attachment of capture probes to features of the array. A “feature” is an entity that acts as a support or repository for various molecular entities used in spatial analysis. In some embodiments, some or all of the features in an array are functionalized for analyte capture. Exemplary substrates are described in Section (II)(c)-69-FH12653613.3of WO 2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663.Exemplary features and geometric attributes of an array can be found in Sections (II)(d)(i), (II)(d)(iii), and (II)(d)(iv) of WO 2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663.

[0334] Generally, analytes and / or intermediate agents (or portions thereof) can be captured when contacting a biological sample with a substrate including capture probes (e.g., a substrate with capture probes embedded, spotted, printed, fabricated on the substrate, or a substrate with features (e.g., beads, wells) including capture probes). As used herein, “contact,” “contacted,” and / or “contacting,” a biological sample with a substrate refers to any contact (e.g., direct or indirect) such that capture probes can interact (e.g., bind covalently or non-covalently (e.g., hybridize)) with analytes from the biological sample. Capture can be achieved actively (e.g., using electrophoresis) or passively (e.g., using diffusion). Analyte capture is further described in Section (II)(e) of WO 2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663.

[0335] In some cases, spatial analysis can be performed by attaching and / or introducing a molecule (e.g., a peptide, a lipid, or a nucleic acid molecule) having a barcode (e.g., a spatial barcode) to a biological sample (e.g., to a cell in a biological sample). In some embodiments, a plurality of molecules (e.g., a plurality of nucleic acid molecules) having a plurality of barcodes (e.g., a plurality of spatial barcodes) are introduced to a biological sample (e.g., to a plurality of cells in a biological sample) for use in spatial analysis. In some embodiments, after attaching and / or introducing a molecule having a barcode to a biological sample, the biological sample can be physically separated (e.g., dissociated) into single cells or cell groups for analysis. Some such methods of spatial analysis are described in Section (III) of WO 2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663.

[0336] In some embodiments, the macromolecular components (e.g., analytes) of individual biological samples (e.g., cells) can be identified or detected with unique identifiers (e.g., barcodes) such that upon characterization of those macromolecular components, such that any given component (e.g., bioanalyte) may be traced to the biological sample (e.g., cell) from which it was obtained. The ability to attribute characteristics to individual biological samples or groups of biological samples is provided by the assignment of unique identifiers specifically to an individual biological sample or groups of biological samples. Unique identifiers, for example, in the form of nucleic acid barcodes, can be assigned or associated -70-FH12653613.3with individual biological samples (e.g., cells) or populations of biological samples (e.g., cells), or genes (e.g., mRNA transcripts, in order to tag or label the biological sample's macromolecular components (and as a result, its characteristics) with the unique identifiers. These unique identifiers can then be used to attribute the biological sample's components and characteristics to an individual biological sample or group of biological samples.

[0337] In some aspects, the unique identifiers are provided in the form of oligonucleotides that include nucleic acid barcode sequences that may be attached to or otherwise associated with the nucleic acid contents of individual biological sample, or to other components of the biological sample, and particularly to fragments of those nucleic acids.

[0338] The nucleic acid barcode sequences can include from 6 to about 20 or more nucleotides within the sequence of the oligonucleotides. In some cases, the length of a barcode sequence may be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, the length of a barcode sequence may be at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, the length of a barcode sequence may be at most 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or shorter. These nucleotides may be completely contiguous, i.e., in a single stretch of adjacent nucleotides, or they may be separated into two or more separate subsequences that are separated by 1 or more nucleotides. In some cases, separated barcode subsequences can be from about 4 to about 16 nucleotides in length. In some cases, the barcode subsequence may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcode subsequence may be at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcode subsequence may be at most 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.

[0339] Moieties (e.g., oligonucleotides) used in the methods described herein can also include other functional sequences useful in processing of nucleic acids from biological samples contained in the droplet. These sequences include, for example, targeted or random / universal amplification primer sequences for amplifying the genomic DNA from the individual biological samples within the droplets while attaching the associated barcode sequences, sequencing primers or primer recognition sites, hybridization or probing sequences, e.g., for identification of presence of the sequences or for pulling down barcoded nucleic acids, or any of a number of other potential functional sequences.-71-FH12653613.3

[0340] The methods described herein may include providing molecular labels, e.g., via a fluid source. The molecular labels may include barcodes (e.g., nucleic acid barcodes). The molecular labels can be provided to the biological sample based on a number of different methods including, without limitation, microinjection, electroporation, liposome-based methods, nanoparticle-based methods, and lipophilic moiety-barcode conjugate methods. For instance, a lipophilic moiety conjugated to a nucleic acid barcode may be contacted with cells or particulate components of interest. The lipophilic moiety may insert into the plasma membrane of a cell thereby labeling the cell with the barcode. The devices and methods of the present disclosure may result in molecular labels being present on (i) the interior of a cell or particulate component and / or (ii) the exterior of a cell or particulate component (e.g., on or within the cell membrane). These and other suitable methods will be appreciated by those skilled in the art (see U.S. Pub. Nos. US20190177800, US20190323088, US20190338353, and US20200002763, each of which is incorporated herein by reference in its entirety).

[0341] In an example, a fluid is provided that includes large numbers of the abovedescribed barcoded oligonucleotides releasably attached to a label. In some cases, a fluid will provide a diverse barcode sequence library that includes at least about 1,000 different barcode sequences, at least about 5,000 different barcode sequences, at least about 10,000 different barcode sequences, at least about 50,000 different barcode sequences, at least about 100,000 different barcode sequences, at least about 1,000,000 different barcode sequences, at least about 5,000,000 different barcode sequences, or at least about 10,000,000 different barcode sequences, or more.

[0342] Oligonucleotides may be releasable from the labels (e.g., optical label, e.g., fluorescent label) upon the application of a particular stimulus. In some cases, the stimulus may be a photo-stimulus, e.g., through cleavage of a photo-labile linkage that releases the oligonucleotides. In other cases, a thermal stimulus may be used, where increase in temperature will result in cleavage of a linkage or other release of the oligonucleotides from the label. In still other cases, a chemical stimulus is used that cleaves a linkage of the oligonucleotides to the label, or otherwise results in release of the oligonucleotides from the label, e.g., beads.

[0343] Methods of Device Manufacture

[0344] The devices of the present disclosure may be fabricated in any of a variety of conventional ways. These structures may be fabricated in whole or in part from polymeric -72-FH12653613.3materials, such as polyethylene or polyethylene derivatives, such as cyclic olefin copolymers (COC), polymethylmethacrylate (PMMA), polydimethylsiloxane (PDMS), polycarbonate, polystyrene, polypropylene, polyvinyl chloride, polytetrafluoroethylene, polyoxymethylene, polyether ether ketone, polycarbonate, polystyrene, or the like, or they may be fabricated in whole or in part from inorganic materials, such as silicon, or other silica based materials, e.g., glass, quartz, fused silica, borosilicate glass, metals, ceramics, and combinations thereof.

[0345] A gasket of the present disclosure may be made in whole or in part from, e.g., a polymer, e.g., a silicone (e.g., silicone rubbers, e.g., PDMS), fluorosilicone, FKM, FFKM, COC elastomer, etc. The gasket may include an elastomeric polymer, e.g., be cut or formed from an elastomeric material, or precursors thereof, e.g., to allow the gasket to be compressible. A gasket may be a composite of compressible and incompressible materials, e.g., an elastomeric polymer bonded to a non-elastomeric polymer, e.g., formed by bonding (e.g., thermally or with adhesive) two or more materials together. Gaskets may include thermoset or thermoplastic polymers, or a combination thereof. A gasket may be coated, e.g., to include a one-sided adhesive, a double-sided adhesive, a polymer coating, or a hydrophobic coating. A hydrophobic coating on the gasket may act to improve sealing (e.g., to prevent leaks), and / or to reduce adhesion between the gasket and the fluidic interface layer and / or substrate layer, e.g., to allow for easier removal.

[0346] A gasket of the disclosure may be formed in place on the fluidic interface layer. For example, a gasket may be printed in place, e.g., using screen printing, CNC controlled nozzle deposition, 3D printing of elastomers on the surface, UV curable processes (e.g., stereolithography), etc. In some embodiments, a gasket may be formed on the fluidic interface by dispensing beads of curable elastomers, e.g., moisture or UV curable elastomers, or, e.g., two-part RTV elastomers. In some embodiments, a gasket may be produced by laser cutting of a continuous gasket layer already laminated on to the substrate layer or fluidic interface layer.

[0347] The fluidic interface layer and substrate layers may be made in whole or in part from glass, polymer (e.g., polystyrene, polycarbonate, polyethylene terephthalate, polypropylene, polyethylene, PTFE, COC, PMMA, etc.), plastic, ceramic, metal, or a combination thereof. The fluidic interface may be constructed of multiple layers, e.g., a top layer and a bottom layer.-73-FH12653613.3

[0348] Polymeric device components may be fabricated using any of a number of processes including soft lithography, embossing techniques, micromachining, e.g., laser machining, or, in some aspects, injection molding of the layer components that include the defined channels as well as other structures, e.g., reservoirs, integrated functional components, etc. In such cases, a laminating layer may be adhered to the molded structured part through readily available methods, including thermal lamination, solvent based lamination, sonic welding, or the like.

[0349] As will be appreciated, structures included of inorganic materials also may be fabricated using known techniques. For example, structures such as channels or reservoirs may be micro-machined into surfaces or etched into the surfaces using standard photolithographic techniques. In some aspects, the devices or components thereof may be fabricated using three-dimensional printing techniques to fabricate the channel or other structures of the devices and / or their discrete components.

[0350] Methods for Surface Modifications

[0351] The disclosure features methods for producing a flow device (e.g., a microfluidic device) that has a surface modification, e.g., a surface with a modified water contact angle. The methods may be employed to modify the surface of a device such that a liquid can “wet” the surface by altering the contact angle the liquid makes with the surface.

[0352] Devices to be modified with surface coating agents may be primed, e.g., pretreated, before coating processes occur. In certain embodiments, the first contact angle is greater than the water contact angle of the primed surface. In other embodiments, the first contact angle is greater than the water contact angle of the device component surface. Thus, the method allows for the differential coating of surfaces within or on the device.

[0353] A surface may be primed by depositing a metal oxide onto it. Example metal oxides useful for priming surfaces include, but are not limited to, A12O3, TiO2, SiO2, or a combination thereof. Other metal oxides useful for surface modifications are known in the art. The metal oxide can be applied to the surface by standard deposition techniques, including, but not limited to, atomic layer deposition (ALD), physical vapor deposition (PVD), e.g., sputtering, chemical vapor deposition (CVD), or laser deposition. Other deposition techniques for coating surfaces, e.g., liquid-based deposition, are known in the art. For example, an atomic layer of A12O3 can be prepared on a surface by depositing trimethylaluminum (TMA) and water.-74-FH12653613.3

[0354] In some cases, the coating agent may create a surface that has a water contact angle greater than 90°, e.g., hydrophobic or fluorophillic, or may create a surface with a water contact angle of less than 90°, e.g., hydrophilic. For example, a fluorophillic surface may be created by flowing fluorosilane (e.g., H3FSi) through a primed device surface, e.g., a surface coated in a metal oxide. The priming of the surfaces of the device enhances the adhesion of the coating agents to the surface by providing appropriate surface functional groups. In some cases, the coating agent used to coat the primed surface may be a liquid reagent.

[0355] While the disclosed subject matter is described herein in terms of certain preferred embodiments, those skilled in the art will recognize that various modifications and improvements may be made to the disclosed subject matter without departing from the scope thereof. Moreover, although individual features of one embodiment of the disclosed subject matter may be discussed herein or shown in the drawings of the one embodiment and not in other embodiments, it should be apparent that individual features of one embodiment may be combined with one or more features of another embodiment or features from a plurality of embodiments.

[0356] In addition to the specific embodiments claimed below, the disclosed subject matter is also directed to other embodiments having any other possible combination of the dependent features claimed below and those disclosed above. As such, the particular features presented in the dependent claims and disclosed above can be combined with each other in other manners within the scope of the disclosed subject matter such that the disclosed subject matter should be recognized as also specifically directed to other embodiments having any other possible combinations. Thus, the foregoing description of specific embodiments of the disclosed subject matter has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosed subject matter to those embodiments disclosed.

[0357] It will be apparent to those skilled in the art that various modifications and variations can be made in the method and system of the disclosed subject matter without departing from the spirit or scope of the disclosed subject matter. Thus, it is intended that the disclosed subject matter include modifications and variations that are within the scope of the appended claims and their equivalents.

[0358] Embodiments-75-FH12653613.3

[0359] The present disclosure provides multiple embodiments. For purpose of illustration and not limitation, some exemplary embodiments, are listed below, and it should be understood by a person of skill in the art that each, or only select, features listed can be combined into a single embodiment, if so desired. Features of exemplary embodiment(s) in accordance with the present disclosure include:1. A system comprising:a moveable stage configured for motion in an X-direction and Y-direction; a pedestal disposed on the moveable stage, the pedestal configured to receive a substrate secured within a cassette, the pedestal having a first side, a second side opposite the first side, and a top surface; andat least one magnetic component disposed relative to the pedestal.2. The system of embodiment 1, comprising a frame configured to support an optical train, wherein the moveable stage is coupled to the frame.3. The system of embodiment 1 or embodiment 2, wherein the first side is a first long side and the second side is a second long side, the pedestal further comprising a first short side and a second short side opposite the second short side.4. The system of any preceding embodiment, wherein the pedestal comprises a first side recess on the first side and a second side recess on the second side.5. The system of any preceding embodiment, wherein the at least one magnetic component comprises a first magnetic component and a second magnetic component.6. The system of embodiment 5, wherein the first magnetic component is disposed within the first side recess and the second magnetic component is disposed within the second side recess.7. The system of any preceding embodiment, wherein the at least one magnetic component is arranged parallel to the first side or the second side.8. The system of any preceding embodiment, wherein the at least one magnetic component in or on the pedestal is arranged to magnetically couple with at least one magnetic component in the cassette.9. The system of any preceding embodiment, wherein the pedestal comprises at least one seating member extending from the top surface and configured to engage the substrate, wherein the seating member has a substantially planar seating surface.-76-FH12653613.310. The system of embodiment 9, wherein the at least one seating member is continuous about a perimeter of the top surface of the pedestal.11. The system of embodiment 9, wherein the at least one seating member is discontinuous about a perimeter of the top surface of the pedestal.12. The system of embodiment 9, wherein the at least one seating member comprises a three- point support.13. The system of embodiment 12, wherein the three-point support comprises a first seating member and a second seating member on a third side of the pedestal and a third seating member on a fourth side of the pedestal.14. The system of any preceding embodiment, wherein the pedestal comprises a chamfer between the first side, the second side, and the top surface, wherein the chamfer is configured to engage with a complementary angled surface on a bottom surface of the cassette.15. The system of any preceding embodiment, wherein the pedestal has a first width from the first side to the second side, and the top surface has a second width that is less than the first width.16. The system of any preceding embodiment, further comprising a cassette presence sensor configured to detect the presence and / or absence of a cassette on the pedestal.17. The system of any preceding embodiment, wherein the at least one magnetic component comprises a ferromagnetic material.18. The system of embodiment 17, wherein the ferromagnetic material comprises iron, alloy steel, stainless steel, nickel, cobalt, gadolinium, neodymium, ferromagnetic ceramic, or a combination thereof.19. The system of any preceding embodiment, further comprising the cassette and the substrate, wherein the substrate includes a sample region configured to receive at least one sample, wherein the cassette comprises a top portion and a bottom portion and the substrate is secured therebetween, and wherein the cassette defines an open well around the sample region.20. A method comprising:positioning a cassette on the pedestal of the system of any one of embodiments 1 to 18 to thereby magnetically couple the at least one magnetic component in or on the pedestal to at least one magnetic component in the cassette, wherein the cassette-77-FH12653613.3comprises a top portion, a bottom portion, and a substrate secured therebetween, wherein the substrate includes a sample region configured to receive at least one sample, and wherein the cassette defines an open well around the sample region; andremoving the cassette from the pedestal.21. The method of embodiment 20, further comprising imaging the at least one sample in the open well after positioning the cassette on the pedestal.22. The method of embodiment 20 or embodiment 21, further comprising a robotic arm configured to position the cassette on the pedestal and / or remove the cassette from the pedestal.23. The method of any one of embodiments 20 to 22, wherein removing the cassette from the pedestal comprises moving the cassette vertically up and away from the pedestal to thereby magnetically decouple the at least one magnetic component in the pedestal from the at least one magnetic component in the pedestal.24. A system comprising:a frame;a moveable stage coupled to the frame and configured for motion in an X- direction and Y-direction;a chuck disposed on the moveable stage, the chuck configured to receive a substrate secured within a cassette casing, the chuck having a first end, a second end, opposing sides, a top surface, and at least one fluidic channel therein configured to apply a vacuum pressure between the sample substrate and the top surface of the chuck;at least one casing registration feature disposed on the moveable stage and configured to align the cassette casing, the at least one casing registration feature including a XY constraint and a X constraint; andan alignment mechanism;wherein the moveable stage is displaced from a first position where the cassette casing is spaced from the casing registration feature to a second position where the cassette casing is engaged with the alignment mechanism and the casing registration feature when the moveable stage translates in at least one of the X-direction and Y- direction.25. The system of embodiment 24, wherein the XY constraint comprises a V-groove.-78-FH12653613.326. The system of embodiment 24 or embodiment 25, wherein the X constraint comprises a substantially flat portion.27. The system of any one of embodiments 24 to 26, wherein the X constraint is offset from the XY constraint by a distance.28. The system of any one of embodiments 24 to 27, wherein the X constraint and the XY constraint are positioned along an axis parallel to a Y axis of the moveable stage.29. The system of any one of embodiments 24 to 28, wherein the alignment mechanism is fixed with respect to the frame.30. The system of embodiment 29, where the alignment mechanism is not on the stage. 31. The system of any one of embodiments 24 to 30, wherein a portion of the alignment mechanism extends in the X-direction and / or Y-direction beyond the moveable stage. 32. The system of any one of embodiments 24 to 31, wherein the alignment mechanism comprises a ball plunger.33. The system of embodiment 32, wherein the ball plunger is aligned perpendicular to the at least one cassette registration feature.34. The system of any one of embodiments 24 to 33, wherein displacing the movable stage from the first position to the second position engages the casing with the alignment mechanism at a midpoint of a side of the casing.35. The system of any one of embodiments 24 to 28, wherein the alignment mechanism is disposed on the moveable stage.36. The system of embodiment 35, wherein the alignment mechanism includes a rotatable cam portion which displaces the alignment mechanism in the X-direction.37. The system of embodiment 36, wherein the rotatable cam portion is coupled to an actuator.38. The system of embodiment 37, wherein the actuator comprises an electric motor.39. The system of embodiment 37, wherein the actuator comprises a pneumatic actuator. 40. The system of any one of embodiments 24 to 39, wherein the first pressure is applied to the chuck during movement of the moveable stage from the first position to the second position.41. The system of any one of embodiments 24 to 40, wherein the chuck includes a heat transfer device.-79-FH12653613.342. The system of any one of embodiments 24 to 41, wherein the at least one casing registration feature is configured to receive at least one kinematic alignment feature disposed on a surface of the cassette casing.43. The system of embodiment 42, wherein each of the at least one kinematic alignment feature of the cassette casing comprises a rigid spherical portion.44. The system of any one of embodiments 24 to 43, wherein displacing the movable stage from the first position to the second position provides an X-force on the cassette casing to thereby register the cassette casing with at least one casing registration feature.45. The system of any one of embodiments 24 to 44, wherein the opposing sides of the chuck include recesses configured to receive tines of a forklift.46. The system of any one of embodiments 24 to 45, wherein the movable stage displaces the casing to align the sample substrate with an imaging device along the Z-axis.47. The system of embodiment 46, wherein the imaging device is at least partially disposed within the casing.48. A method comprising:providing the system of any one of embodiments 24 to 47 ;positioning the substrate on the chuck, wherein the substrate is secured within the cassette casing, wherein the cassette casing comprises at least one kinematic alignment feature disposed on a surface of the cassette casing; andactuating the moveable stage and / or the alignment mechanism to thereby align the at least one kinematic alignment feature with the at least one casing registration feature.49. The method of embodiment 48, further comprising applying a first pressure to the chuck when the cassette casing is disposed thereon; andapplying a second pressure when the moveable stage is in the second position.50. The method of embodiment 49, wherein the first pressure is less than the second pressure, and the second pressure is configured to maintain the substrate in a substantially parallel orientation with an XY plane defined by an upper surface of the chuck.51. A method comprising:providing a system comprising:a frame;a moveable stage coupled to the frame and configured for motion in an -80-FH12653613.3X-direction and Y-direction;a chuck disposed on the moveable stage, the chuck configured to receive a substrate secured within a cassette casing, the chuck having a first end, a second end, opposing sides, a top surface, and at least one fluidic channel therein configured to apply a vacuum pressure between the sample substrate and the top surface of the chuck;at least one casing registration feature disposed on the moveable stage and configured to align the cassette casing, the at least one casing registration feature including a XY constraint and a X constraint;an alignment mechanism positioned on the frame, wherein the alignment mechanism is fixed with respect to the frame and extends in the X-direction and / or Y-direction beyond the moveable stage;positioning the substrate on the chuck, wherein the substrate is secured within the cassette casing, wherein the cassette casing comprises at least one kinematic alignment feature disposed on a surface of the cassette casing; andactuating the moveable stage from a first position where the cassette casing is spaced from the casing registration feature to a second position where the cassette casing is engaged with the alignment mechanism and the at least one kinematic alignment feature is engaged with the at least one casing registration feature.52. The system of embodiment 51, wherein the alignment mechanism comprises a ball plunger aligned perpendicular to the at least one cassette registration feature.53. The system of embodiment 51 or embodiment 52, further comprising applying a first pressure to the chuck when the cassette casing is disposed thereon; andapplying a second pressure when the moveable stage is in the second position.54. The system of embodiment 53, wherein the first pressure is less than the second pressure, and the second pressure is configured to maintain the substrate in a substantially parallel orientation with an XY plane defined by an upper surface of the chuck.-81-FH12653613.3

Claims

CLAIMS1. A system comprising:a moveable stage configured for motion in an X-direction and Y-direction; a pedestal disposed on the moveable stage, the pedestal configured to receive a substrate secured within a cassette, the pedestal having a first side, a second side opposite the first side, and a top surface; andat least one magnetic component disposed relative to the pedestal.

2. The system of claim 1, comprising a frame configured to support an optical train, wherein the moveable stage is coupled to the frame.

3. The system of claim 1 or claim 2, wherein the first side is a first long side and the second side is a second long side, the pedestal further comprising a first short side and a second short side opposite the second short side.

4. The system of any preceding claim, wherein the pedestal comprises a first side recess on the first side and a second side recess on the second side.

5. The system of any preceding claim, wherein the at least one magnetic component comprises a first magnetic component and a second magnetic component.

6. The system of claim 5, wherein the first magnetic component is disposed within the first side recess and the second magnetic component is disposed within the second side recess.

7. The system of any preceding claim, wherein the at least one magnetic component is arranged parallel to the first side or the second side.

8. The system of any preceding claim, wherein the at least one magnetic component in or on the pedestal is arranged to magnetically couple with at least one magnetic component in the cassette when the cassette is positioned on the pedestal.

9. The system of any preceding claim, wherein the pedestal comprises at least one seating member extending from the top surface and configured to engage the substrate, wherein the seating member has a substantially planar seating surface.

10. The system of claim 9, wherein the at least one seating member is continuous about a perimeter of the top surface of the pedestal.

11. The system of claim 9, wherein the at least one seating member is discontinuous about a perimeter of the top surface of the pedestal.-82-FH12653613.

312. The system of claim 9, wherein the at least one seating member comprises a three- point support.

13. The system of claim 12, wherein the three-point support comprises a first seating member and a second seating member on a third side of the pedestal and a third seating member on a fourth side of the pedestal.

14. The system of any preceding claim, wherein the pedestal comprises a chamfer between the first side, the second side, and the top surface, wherein the chamfer is configured to engage with a complementary angled surface on a bottom surface of the cassette.

15. The system of any preceding claim, wherein the pedestal has a first width from the first side to the second side, and the top surface has a second width that is less than the first width.

16. The system of any preceding claim, further comprising a cassette presence sensor configured to detect the presence and / or absence of a cassette on the pedestal.

17. The system of any preceding claim, wherein the at least one magnetic component comprises a ferromagnetic material.

18. The system of claim 17, wherein the ferromagnetic material comprises iron, alloy steel, stainless steel, nickel, cobalt, gadolinium, neodymium, ferromagnetic ceramic, or a combination thereof.

19. The system of any preceding claim, further comprising the cassette and the substrate, wherein the substrate includes a sample region configured to receive at least one sample, wherein the cassette comprises a top portion and a bottom portion and the substrate is secured therebetween, and wherein the cassette defines an open well around the sample region.

20. A method comprising:positioning a cassette on the pedestal of the system of any one of claims 1 to 18 to thereby magnetically couple the at least one magnetic component in or on the pedestal to at least one magnetic component in the cassette, wherein the cassette comprises a top portion, a bottom portion, and a substrate secured therebetween, wherein the substrate includes a sample region configured to receive at least one sample, and wherein the cassette defines an open well around the sample region; and removing the cassette from the pedestal.-83-FH12653613.

321. The method of claim 20, further comprising imaging the at least one sample in the open well after positioning the cassette on the pedestal.

22. The method of claim 20 or claim 21, further comprising a robotic arm configured to position the cassette on the pedestal and / or remove the cassette from the pedestal.

23. The method of any one of claims 20 to 22, wherein removing the cassette from the pedestal comprises moving the cassette vertically up and away from the pedestal to thereby magnetically decouple the at least one magnetic component in the pedestal from the at least one magnetic component in the pedestal.

24. A system comprising:a frame;a moveable stage coupled to the frame and configured for motion in an X- direction and Y-direction;a chuck disposed on the moveable stage, the chuck configured to receive a substrate secured within a cassette casing, the chuck having a first end, a second end, opposing sides, a top surface, and at least one fluidic channel therein configured to apply a vacuum pressure between the sample substrate and the top surface of the chuck;at least one casing registration feature disposed on the moveable stage and configured to align the cassette casing, the at least one casing registration feature including a XY constraint and a X constraint; andan alignment mechanism;wherein the moveable stage is displaced from a first position, in which the cassette casing is spaced from the casing registration feature, to a second position, in which the cassette casing is engaged with the alignment mechanism and the casing registration feature, when the moveable stage translates in at least one of the X- direction and Y-direction.

25. The system of claim 24, wherein the XY constraint comprises a V-groove.

26. The system of claim 24 or claim 25, wherein the X constraint comprises a substantially flat portion.

27. The system of any one of claims 24 to 26, wherein the X constraint is offset from the XY constraint by a distance.-84-FH12653613.

328. The system of any one of claims 24 to 27, wherein the X constraint and the XY constraint are positioned along an axis parallel to a Y axis of the moveable stage.

29. The system of any one of claims 24 to 28, wherein the alignment mechanism is fixed with respect to the frame.

30. The system of claim 29, where the alignment mechanism is not on the stage.

31. The system of any one of claims 24 to 30, wherein a portion of the alignment mechanism extends in the X-direction and / or Y-direction beyond the moveable stage.

32. The system of any one of claims 24 to 31, wherein the alignment mechanism comprises a ball plunger.

33. The system of claim 32, wherein the ball plunger is aligned perpendicular to the at least one cassette registration feature.

34. The system of any one of claims 24 to 33, wherein displacing the movable stage from the first position to the second position engages the casing with the alignment mechanism at a midpoint of a side of the casing.

35. The system of any one of claims 24 to 28, wherein the alignment mechanism is disposed on the moveable stage.

36. The system of claim 35, wherein the alignment mechanism includes a rotatable cam portion which displaces the alignment mechanism in the X-direction.

37. The system of claim 36, wherein the rotatable cam portion is coupled to an actuator.

38. The system of claim 37, wherein the actuator comprises an electric motor.

39. The system of claim 37, wherein the actuator comprises a pneumatic actuator.

40. The system of any one of claims 24 to 39, wherein the first pressure is applied to the chuck during movement of the moveable stage from the first position to the second position.

41. The system of any one of claims 24 to 40, wherein the chuck includes a heat transfer device.

42. The system of any one of claims 24 to 41, wherein the at least one casing registration feature is configured to receive at least one kinematic alignment feature disposed on a surface of the cassette casing.

43. The system of claim 42, wherein each of the at least one kinematic alignment feature of the cassette casing comprises a rigid spherical portion.-85-FH12653613.

344. The system of any one of claims 24 to 43, wherein displacing the movable stage from the first position to the second position provides an X-force on the cassette casing to thereby register the cassette casing with at least one casing registration feature.

45. The system of any one of claims 24 to 44, wherein the opposing sides of the chuck include recesses configured to receive tines of a forklift.

46. The system of any one of claims 24 to 45, wherein the movable stage displaces the casing to align the sample substrate with an imaging device along the Z-axis.

47. The system of claim 46, wherein the imaging device is at least partially disposed within the casing.

48. A method comprising:providing the system of any one of claims 24 to 47 ;positioning the substrate on the chuck, wherein the substrate is secured within the cassette casing, wherein the cassette casing comprises at least one kinematic alignment feature disposed on a surface of the cassette casing; andactuating the moveable stage and / or the alignment mechanism to thereby align the at least one kinematic alignment feature with the at least one casing registration feature.

49. The method of claim 48, further comprising applying a first pressure to the chuck when the cassette casing is disposed thereon; andapplying a second pressure when the moveable stage is in the second position.

50. The method of claim 49, wherein the first pressure is less than the second pressure, and the second pressure is configured to maintain the substrate in a substantially parallel orientation with an XY plane defined by an upper surface of the chuck.

51. A method comprising:providing a system comprising:a frame;a moveable stage coupled to the frame and configured for motion in an X-direction and Y-direction;a chuck disposed on the moveable stage, the chuck configured to receive a substrate secured within a cassette casing, the chuck having a first end, a second end, opposing sides, a top surface, and at least one fluidic channel therein configured to apply a vacuum pressure between the sample substrate and the top -86-FH12653613.3surface of the chuck;at least one casing registration feature disposed on the moveable stage and configured to align the cassette casing, the at least one casing registration feature including a XY constraint and a X constraint;an alignment mechanism positioned on the frame, wherein the alignment mechanism is fixed with respect to the frame and extends in the X-direction and / or Y-direction beyond the moveable stage;positioning the substrate on the chuck, wherein the substrate is secured within the cassette casing, wherein the cassette casing comprises at least one kinematic alignment feature disposed on a surface of the cassette casing; andactuating the moveable stage from a first position where the cassette casing is spaced from the casing registration feature to a second position where the cassette casing is engaged with the alignment mechanism and the at least one kinematic alignment feature is engaged with the at least one casing registration feature.

52. The system of claim 51, wherein the alignment mechanism comprises a ball plunger aligned perpendicular to the at least one cassette registration feature.

53. The system of claim 51 or claim 52, further comprising applying a first pressure to the chuck when the cassette casing is disposed thereon; andapplying a second pressure when the moveable stage is in the second position.

54. The system of claim 53, wherein the first pressure is less than the second pressure, and the second pressure is configured to maintain the substrate in a substantially parallel orientation with an XY plane defined by an upper surface of the chuck.-87-FH12653613.3

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