System including a biological sample processing chamber

Abstract

The present disclosure relates to a sample processing assembly, which includes (a) a lower plate (10), (b) an upper plate (30) complementary to the lower plate, and (c) a chamber formed by them. In some embodiments, the formed chamber is suitable for performing demasking operations, such as antigen repair and / or target repair. In some embodiments, the sample processing assembly is configured to keep the sample-carrying substrate (15) horizontal during all processing steps. The present disclosure also relates to a system including one or more independently operable sample processing assemblies.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This disclosure claims the benefit of the filing date of U.S. Provisional Application No. 62 / 891,118, filed on August 23, 2019; and U.S. Provisional Application No. 62 / 847,388, filed on May 14, 2019, the disclosures of which are incorporated herein by reference in their entireties. Technical Field

[0003] The present disclosure relates to a system for unmasking protein antigens and nucleic acid targets from fixed biological samples. Background Art

[0004] Fixation of tissue and cell samples is used to help ensure that the morphology of the sample and the spatial distribution of biomolecules are preserved, and thus enable pathologists to make a diagnosis. As an adjunct to morphological indicators of disease, the presence of specific protein and / or nucleic acid sequences can be used to further characterize the disease state and, in some cases, be used by oncologists to guide treatment of the disease. However, fixation can also interfere with the detection of proteins and nucleic acids in the sample. Therefore, for immunohistochemistry (IHC) procedures and In situ Hybridization (ISH) procedures typically use a unmasking step (also called "antigen retrieval" or "target retrieval" for IHC and ISH, respectively) to make protein antigens or nucleic acid targets accessible to detection reagents (such as antibodies or probes).

[0005] Sample fixation is typically accomplished using neutral buffered formalin (NBF). The formaldehyde in NBF is believed to preserve tissue and cell morphology by forming crosslinks between reactive groups on proteins and nucleic acids in the sample, and these crosslinks can render certain portions of the molecules undetectable. For example, formaldehyde primarily preserves or fixes tissues and cells by crosslinking primary amine groups in proteins to other nearby nitrogen atoms in proteins or DNA through -CH2- bonds. However, tissue fixation methods often mask antigens on specific proteins that are required for detection for diagnostic and prognostic purposes. Various methods are used to reverse the effects of formalin fixation and access antigens and targets in fixed biological samples. However, none of these methods is considered completely successful in all cases, and some are more amenable to automation than others. Summary of the Invention

[0006] The present disclosure relates to a sample processing assembly comprising (a) a lower plate and (b) an upper plate that is complementary to the lower plate. The "complementary" upper and lower plates each include features or sets of features that complement each other. For example, the lower plate may have a first set of features (e.g., a substrate table and a lower engagement surface), and the upper plate may have a second set of features (e.g., a cavity and an upper engagement surface), wherein the first set of features and the second set of features complement each other. In some embodiments, the sample processing assembly includes independently movable complementary lower and upper plates. For example, the lower plate may be coupled to a subassembly that is movable toward the upper plate (e.g., a fixed upper plate or a movable upper plate). In another example, the upper plate may be coupled to a subassembly that is movable toward the lower plate (e.g., a fixed lower plate or a movable lower plate). In some embodiments, the lower plate has a modular design (a "modular lower plate"), which allows the lower plate to be used with a variety of different sample processing assemblies.

[0007] In some embodiments, the sample processing assembly comprises a chamber formed by complementary upper and lower plates (or a modular lower plate having features complementary to the upper plate). In some embodiments, the sample processing assembly is suitable for demasking a sample disposed on a substrate and provided within the chamber, such as for antigen retrieval and / or target retrieval. In some embodiments, during the demasking operation, at least a portion of the sample disposed on the substrate and / or the substrate itself is maintained at a temperature lower than any other component within the chamber. For example, the sample disposed on the substrate or the portion of the substrate itself can be maintained as the "coldest" component within the chamber during the demasking operation. Other aspects of the sample processing assembly and components of such sample processing assemblies are further described herein. The present disclosure also relates to systems comprising one or more independently operable sample processing assemblies.

[0008] In one aspect of the present disclosure, a sample processing assembly includes (a) a lower plate and (b) an upper plate complementary to the lower plate. In some embodiments, the lower plate and the upper plate have complementary polygonal shapes. In some embodiments, the lower plate and the upper plate have complementary wedge-like shapes. In some embodiments, at least one of the lower plate and the upper plate is movable. In some embodiments, both the lower plate and the upper plate are independently movable.

[0009] In some embodiments, the lower plate includes a first set of features and the upper plate includes a second set of features, wherein the first set of features and the second set of features are complementary to each other. In some embodiments, the first set of features includes a lower engagement surface and one or more substrate tables. In some embodiments, the one or more substrate tables are elevated relative to at least a portion of the lower engagement surface. In some embodiments, the one or more substrate tables are recessed relative to at least a portion of the lower engagement surface. In some embodiments, the second set of features includes an upper engagement surface. In some embodiments, the second set of features includes the upper engagement surface and one or more cavities. In some embodiments, the one or more cavities are recessed relative to at least a portion of the upper engagement surface. In some embodiments, each of the lower and upper plates includes additional features, including one or more ports, one or more heating elements, one or more cooling elements, one or more substrate alignment members, and the like. In some embodiments, the one or more ports may be gas inlets, through which one or more gases and / or vapors may be supplied. In some embodiments, the one or more ports may be exhaust ports, through which gases and / or vapors may be released.

[0010] In some embodiments, the lower plate has a modular design. In some embodiments, the modular lower plate includes a main body having a lower engagement surface. In some embodiments, the modular lower plate includes a thermal management module. In some embodiments, the main body can rest on, engage with, or be coupled to the thermal management module. In some embodiments, the main body includes a substrate table that is integral with and integral to the main body. In some embodiments, when a substrate is supported by the substrate table of the main body (wherein the main body, including the substrate table and thermal management module, together constitutes the modular lower plate), the main body can be picked up, transported, and deposited onto the thermal management module. In some embodiments, the main body is contained within a carrier block. In some embodiments, the carrier block and the main body can be picked up, transported, and deposited onto the thermal management module. In other embodiments, the main body includes a lower engagement surface but does not include an integral substrate table. Conversely, the modular lower plate includes a main body having a detachable substrate table. In this manner, the detachable substrate table can serve as a carrier for the substrate. For example, the substrate can be placed on the detachable substrate table, and the substrate and detachable substrate table can be picked up, transported, and deposited onto the main body together. In some embodiments, the substrate and separate substrate stage pair remain together throughout all or at least some of the processing steps that a sample disposed on the substrate may undergo, for example, all or part of the steps used to prepare a sample for microscopic analysis, such as from baking to coverslipping or from baking to staining. In some embodiments, a user places a substrate bearing a sample on a separate substrate stage and inputs the substrate / substrate stage pair into the system and, after sample processing, retrieves the substrate / substrate stage from the system and then removes the substrate with the processed sample thereon for analysis.

[0011] In some embodiments, the complementary upper plate and lower plate include complementary upper and lower engaging surfaces, respectively. In some embodiments, both the complementary upper and lower engaging surfaces include complementary flat surfaces. In some embodiments, both the complementary upper and lower engaging surfaces include complex complementary surfaces, such as complementary surfaces including curves or arcuate shapes. In some embodiments, the complementary upper and lower engaging surfaces are configured so that a sealed joint can be formed when the upper and lower engaging surfaces at least partially contact each other or contact a sealing body disposed therebetween. In some embodiments, the sealed joint between the complementary upper and lower engaging surfaces facilitates the formation of a sealed chamber.

[0012] In some embodiments, the sealed chamber is heated and / or pressurized for a predetermined amount of time and then cooled. In some embodiments, the sealed chamber is heated and / or pressurized for a predetermined amount of time and then opened without first cooling. In some embodiments, during the demasking operation, at least a portion of the sample disposed on the substrate and / or at least a portion of the substrate itself is maintained at a temperature lower than any other component within the sealed chamber. For example, during all steps of the demasking operation, such as during heating, pressurization, cooling, decompression, quenching, dispensing additional fluid, etc., the sample disposed on the substrate or the substrate itself can be maintained as the "coldest" component within the sealed chamber. For example, the sample and / or the portion of the substrate may be maintained at a temperature that is at least about 2°C lower than any other component within the sealed chamber. For another example, the sample and / or the portion of the substrate may be maintained at a temperature that is at least about 5°C lower than any other component within the sealed chamber.

[0013] In some embodiments, any fluids and / or reagents required to perform the demasking operation are first introduced to the sample, a portion of the substrate itself, and / or a reservoir disposed on the substrate, and then the chamber is sealed. For example, before forming the sealed chamber between the upper and lower plates, a total volume of one or more fluids and / or reagents ranging from about 250 μL to about 1000 μL can be dispensed into the sample, a portion of the substrate itself, and / or a reservoir within the lower plate. In some embodiments, no additional fluids and / or reagents are applied after the sealed chamber is formed.

[0014] In some embodiments, as part of the demasking operation, one or more additional fluids and / or reagents are dispensed into the sample and / or substrate in the sealed chamber. For example, as part of the demasking operation, between about 250 μL and about 1000 μL of one or more additional fluids and / or reagents may be dispensed into the sealed chamber. In some embodiments, as part of the demasking operation, one or more additional fluids and / or reagents are dispensed into the sealed chamber such that at least about 90% of the volume of the chamber is filled with fluids and / or reagents. In some embodiments, as part of the demasking operation, one or more additional fluids and / or reagents are dispensed into the sealed chamber such that at least about 95% of the volume of the chamber is filled with fluids and / or reagents. In some embodiments, as part of the demasking operation, one or more additional fluids and / or reagents are dispensed into the sealed chamber such that at least about 99% of the volume of the chamber is filled with fluids and / or reagents.

[0015] In some embodiments, the body of the lower plate or the one or more substrate tables themselves are configured such that the upper surface of the substrate table is horizontal, e.g., parallel to the ground. In those embodiments where the lower plate is movable, the one or more substrate tables and / or the lower body are configured such that the substrate supported by the upper surface remains horizontal as the lower plate moves. In some embodiments, the upper surface of the substrate table remains horizontal throughout the movement of the lower plate, regardless of whether the movement of the lower plate remains completely parallel to the ground or whether there is a vertical component to the movement of the lower plate (including those embodiments where there is a completely vertical movement). In embodiments where the bodies of the lower and upper plates have complementary wedge-based shapes, the elevated substrate table itself can have a substantially wedge-based shape such that the upper surface of the elevated substrate table remains horizontal as the lower plate moves.

[0016] In some embodiments, the complementary lower and upper plates are independently movable so that they contact each other at the interface of the complementary lower and upper engaging surfaces. In some embodiments, the movement of either the upper and lower plates is affected by one or more of a motor, a screw, a lever, a spring, a cam mechanism, a piston, or any combination thereof. In some embodiments, after the plates are at least partially in contact with each other, an additional external force is applied to one or both of the upper and / or lower plates. In some embodiments, applying the additional external force is beneficial in maintaining a sealed joint between the upper and lower plates (with or without a sealing body, as described below). In some embodiments, the external force is applied using one or more force generating components (e.g., one or more of a motor, a piston, a spring, a screw mechanism, a lever, and / or a cam mechanism).

[0017] In some embodiments, both the lower plate and the upper plate are simultaneously moved toward each other until they contact one another (e.g., their respective upper and lower engagement surfaces contact one another), and then one or more force-generating members are engaged to exert additional force on at least the upper plate. In some embodiments, one or more force-generating members in communication with at least the upper plate may be engaged to urge the upper plate against the lower plate, thereby further promoting a sealing engagement between the upper and lower plates or enabling the sealing engagement to be maintained when internal pressure in the chamber formed by the upper and lower plates increases (e.g., when internal pressure increases during a demasking operation and / or due to pre-pressurization).

[0018] In some embodiments, each of the lower plate and the upper plate can be independently movable in any one of the x-coordinate direction, the y-coordinate direction, and the z-coordinate direction. In some embodiments, both the lower plate and the upper plate can be simultaneously movable toward each other in any one of the x-coordinate direction, the y-coordinate direction, and the z-coordinate direction. In some embodiments, one of the upper plate or the lower plate can be moved to a predetermined position, and simultaneously or subsequently, the other can be moved toward the positioned plate. In some embodiments, the lower plate can be moved to a predetermined position, and then, simultaneously or subsequently, the upper plate can be moved toward the positioned lower plate. In some embodiments, the lower plate is moved to a predetermined position, and the upper plate can be moved downward toward the lower plate.

[0019] In some embodiments, one of the lower plate or the upper plate is movable and the other of the lower plate or the upper plate is immovable. In some embodiments, the upper plate is fixed and the lower plate is movable toward the upper plate. In some embodiments, the lower plate is fixed and the upper plate is movable toward the lower plate.

[0020] In some embodiments, the lower plate moves from the loading area to at least one of the preparation area or the demasking area. In some embodiments, the movement of the lower plate facilitates the treatment of the substrate supported by the lower plate with one or more fluids and / or reagents. In some embodiments, the lower plate is fixed and one or more dispensing devices move to the fixed lower plate to dispense one or more fluids and / or reagents to the substrate supported by the lower plate. For example, before forming a sealed chamber between the lower plate and the upper plate, one or more fluids and / or reagents with a total volume of between about 250 μL and about 1000 μL can be dispensed to a sample disposed on the substrate, a portion of the substrate itself, and / or a reservoir within the lower plate. In some embodiments, once the substrate is positioned within the sealed chamber, no additional fluids and / or reagents are dispensed to the substrate.

[0021] In some embodiments, the complementary lower and upper engaging surfaces facilitate sealing engagement without the use of any sealing body disposed therebetween. In some embodiments, at least one of the lower plate and the upper plate comprises at least one sealing body. In some embodiments, the sealing body may be disposed in a groove of the complementary upper and / or lower plate, and the sealing body together with the complementary upper and lower engaging surfaces facilitate sealing engagement between the upper and lower plates. In some embodiments, the at least one sealing body is removable. In some embodiments, the sealing body may be disposed between the complementary upper and lower engaging surfaces without being disposed in a groove. In some embodiments, the sealing body is positioned on the surface of the lower engaging surface. In these embodiments, when the upper plate is brought into contact with the sealing element positioned on the lower engaging surface, the sealing engagement can be maintained, for example, by applying an external force to the lower and / or upper plates with one or more force generating members. In some embodiments, the at least one removable seal is integrated in a removable sealing attachment, wherein the removable sealing attachment is configured to engage a portion of the periphery of the lower or upper plate. In some embodiments, one of the complementary lower joining surfaces or upper joining surfaces includes a raised sealing member protruding from a plane formed by the one of the complementary lower joining surfaces or upper joining surfaces, and wherein the other of the complementary lower joining surfaces or upper joining surfaces includes a channel complementary to the raised sealing member.

[0022] In some embodiments, at least one of the upper plate and the lower plate further comprises one or more heating and / or cooling elements. In some embodiments, the cooling element is an active cooling element. In some embodiments, the active cooling element comprises a tube in at least partial contact with at least one of the body of the lower plate or the body of the upper plate, wherein the tube is connected to a circulation device (e.g., a refrigerator) so that a liquid heat transfer medium can be circulated through the tube to achieve cooling of at least a portion of the lower plate, the upper plate, and / or the substrate disposed on the substrate table. In some embodiments, the cooling element is a passive cooling element. In some embodiments, the passive cooling element is a radiator. In some embodiments, only the upper plate comprises a heating element. In some embodiments, at least one of the upper plate or the lower plate comprises a heating element, and wherein the lower plate comprises a passive or active cooling element. In some embodiments, only the lower plate comprises a heating element.

[0023] In embodiments where both the upper and lower plates include heating and / or cooling elements, the heating and / or cooling elements can be operated independently, as described herein. In some embodiments, any of the heating and / or cooling elements within the upper plate can be operated in conjunction with those within the lower plate or those embedded within one or more thermal management modules. Consequently, by independently controlling the various heating and / or cooling elements present in the upper and lower plates, the temperature of the substrate table, any portion of the substrate supported by the substrate table, and / or the chamber can be controlled. In some embodiments, independent control of the various heating and / or cooling elements enables the creation and maintenance of a thermal gradient, such as between the substrate supported by the substrate table and the walls within the chamber.

[0024] In some embodiments, at least one of the upper plate and the lower plate further includes a sensor, such as a temperature sensor. In some embodiments, the upper plate includes a temperature sensor that contacts the substrate and / or the sample disposed on the substrate. In some embodiments, the lower plate includes a temperature sensor that contacts the substrate. In some embodiments, data from the temperature sensor is monitored so that one or more heating and / or cooling elements disposed in the lower plate and / or the upper plate can be controlled. For example, a sensor can provide feedback so that the one or more heating and / or cooling elements disposed in the lower plate and / or the upper plate can be controlled to keep a portion of the sample or substrate as the coldest component in the sealed chamber.

[0025] Another aspect of the present disclosure is a sample processing assembly comprising: (i) a lower plate coupled to a subassembly, wherein the lower plate comprises a lower engagement surface and one or more substrate tables, wherein the one or more substrate tables comprise an upper surface adapted to horizontally hold a substrate; and (ii) an upper plate having an upper engagement surface complementary to the lower engagement surface. In some embodiments, the upper plate further comprises one or more cavities. In some embodiments, the one or more cavities each receive at least a portion of the one or more substrate tables. In some embodiments, the lower plate and one of the one or more substrate tables are configured such that the upper surface of the one or more substrate tables remains horizontal as the lower plate traverses the subassembly (e.g., any substrate supported by the one or more substrate tables remains parallel to the ground throughout movement of the lower plate along the subassembly, regardless of whether the movement is entirely horizontal along the subassembly or includes a vertical component of movement, or even purely vertical movement). In some embodiments, the substrate is a microscope slide.

[0026] In some embodiments, the lower plate is coupled to a subassembly that is configured to move the lower plate into contact with the upper plate. In some embodiments, the subassembly is moved with sufficient force such that the movement of the subassembly itself is sufficient to form a seal between the lower plate coupled to the subassembly and the upper plate. For example, the subassembly may include a device for translating the lower plate along a track (e.g., a horizontally arranged track or a track with one end offset from the horizontal, as described below). In some embodiments, the subassembly includes a motor adapted to move the lower plate along the track, e.g., from a loading position to an unmasking position.

[0027] In some embodiments, the subassembly is arranged horizontally. In this configuration, the lower plate moves along the horizontally arranged subassembly. In some embodiments, the subassembly is arranged so that the first end of the subassembly is elevated vertically relative to the second end of the subassembly, e.g., the subassembly is offset horizontally. In this particular embodiment, when the lower plate moves along the subassembly, it does so with a motion that includes a vertical component. For example, when the lower plate moves in either the x-direction and / or the y-direction of the horizontally offset subassembly, there is concomitant movement in the z-direction given the horizontal offset of the track. In some embodiments, the track is offset from the horizontal by an angle ranging from approximately 5 degrees to approximately 70 degrees. In other embodiments, the horizontal offset angle ranges from approximately 5 degrees to approximately 60 degrees. In yet other embodiments, the horizontal offset angle ranges from approximately 10 degrees to approximately 50 degrees. In further embodiments, the horizontal offset angle ranges from approximately 20 degrees to approximately 50 degrees. In yet other embodiments, the horizontal offset angle ranges from approximately 20 degrees to approximately 45 degrees. In some embodiments, the subassembly further includes a motor adapted to move the lower plate along the subassembly.

[0028] In some embodiments, the upper plate is coupled to the support member and the lower plate is moved (along a horizontal subassembly or a horizontally offset subassembly) to a position where the lower engagement surface contacts a complementary upper engagement surface of the upper plate. In some embodiments, the upper plate is coupled to one or more springs and the lower plate is moved to a position where the lower engagement surface contacts a complementary upper engagement surface of the upper plate, and wherein the one or more springs exert a downward force on the upper plate when the lower plate contacts the upper plate.

[0029] In some embodiments, the lower plate moves to a predetermined position below the upper plate, and the upper plate moves toward the positioned lower plate at least along the z-axis. In other embodiments, the lower plate moves closer to the upper plate, and the upper plate simultaneously or subsequently moves toward the lower plate (in any of the x-, y-, and z-directions) until the upper engaging surface of the upper plate contacts the lower engaging surface of the lower plate. In still other embodiments, both the lower and upper plates move toward each other simultaneously (herein, the movement of each of the lower and upper plates can be independent in any of the x-, y-, and / or z-directions). In some embodiments, both the lower and upper plates move toward each other simultaneously until they contact each other (e.g., their respective upper and lower engaging surfaces contact each other), and then engage a force-generating member connected to the upper plate. In some embodiments, the engagement of the force-generating member causes a force to be applied to at least the upper plate. In some embodiments, the force applied to the upper plate facilitates a sealed engagement between the upper and lower plates. In some embodiments, a predetermined amount of force is applied to the upper plate by the force-generating member. In some embodiments, the predetermined amount of force applied by the force generating member to the upper plate is greater than the force generated by pressurizing the interior of the chamber formed by the upper and lower plates, but is below a predetermined threshold force above which an unsafe pressure may be generated within the chamber. In some embodiments, this predetermined force applied by the force generating member is limited such that if the pressure within the chamber exceeds the predetermined pressure, the force applied by the force generating member is overcome and the force generating member will slide or retract to relieve pressure that may be generated within the chamber above the predetermined pressure.

[0030] In some embodiments, the sample processing assembly includes a chamber. In some embodiments, the chamber is formed by a complementary lower plate and an upper plate. In some embodiments, the complementary lower engaging surface and the upper engaging surface of the lower plate and the upper plate are respectively conducive to the sealing engagement between the upper plate and the lower plate, thereby forming a chamber therebetween. In some embodiments, the complementary lower engaging surface and the upper engaging surface provide sealing engagement without using any sealing body arranged therebetween. In some embodiments, the sealing body can be arranged in the groove of the upper plate and / or the lower plate, and the sealing body together with the upper engaging surface and the lower engaging surface are conducive to the sealing engagement between the upper plate and the lower plate. In some embodiments, the sealing body can be arranged between the upper engaging surface and the lower engaging surface and is not arranged in, for example, a groove, for example, the sealing body rests on the surface of the lower engaging surface. In some embodiments, one of the lower engaging surface or the upper engaging surface comprises a raised sealing member protruding from the plane formed by the lower engaging surface or the upper engaging surface, and wherein the other of the lower engaging surface or the upper engaging surface comprises a channel complementary to the raised sealing member.

[0031] In some embodiments, one or both of the lower plate and the upper plate include one or more heating and / or cooling elements. In some embodiments, the one or more heating and / or cooling elements within the lower and upper plates operate together to establish and maintain a predetermined thermal gradient within the chamber, for example. For example, the one or more heating and / or cooling elements within the lower and upper plates operate together to establish and maintain a thermal gradient between the substrate table and other components within the chamber, such as the walls defining the chamber interior. In some embodiments, the one or more heating and / or cooling elements are independently controlled to maintain a sample disposed on a substrate and / or a portion of the substrate itself at a lower temperature than any other components within the chamber (e.g., chamber walls, ports, valves, sensors, probes, etc.). For example, the sample and / or portion of the substrate can be maintained at a temperature at least approximately 2°C cooler than any other components within the sealed chamber. As another example, the sample and / or portion of the substrate can be maintained at a temperature at least approximately 5°C cooler than any other components within the sealed chamber. In some embodiments, the upper plate further includes one or more ports. In some embodiments, the one or more ports allow for the introduction of one or more gases and / or vapors into the chamber formed by the lower and upper plates. For example, steam can be introduced to heat the sample in the chamber and simultaneously pressurize the chamber. In some embodiments, the upper plate further includes one or more valves to facilitate the release of gas and / or steam from the chamber formed by the lower plate and the upper plate. For example, one or more valves can be opened for a predetermined amount of time so that pressure can be released from the sealed chamber. Alternatively, the sealed chamber can be opened immediately after the demasking operation is completed, for example, the sealed chamber can be opened without first cooling or depressurizing the chamber.

[0032] In some embodiments, the demasking operation performed within the chamber formed by the complementary lower and upper plates is performed using fluids and / or reagents supplied to the substrate prior to chamber formation. In some embodiments, no additional fluids and / or reagents are dispensed to the substrate while the substrate is positioned within the chamber and after the chamber is sealed.

[0033] In some embodiments, the chamber may include one or more ports, such as through which one or more additional fluids and / or reagents may be added directly into the sealed chamber as part of a demasking operation. In some embodiments, as part of a demasking operation, one or more additional fluids and / or reagents are dispensed into the sample and / or substrate in the sealed chamber. For example, as part of a demasking operation, between about 250 μL and about 1000 μL of one or more additional fluids and / or reagents may be dispensed into the sealed chamber. In some embodiments, as part of a demasking operation, one or more additional fluids and / or reagents are dispensed into the sealed chamber such that at least about 90% of the volume of the chamber is filled with fluids and / or reagents. In some embodiments, the lower plate may include one or more ports, such as vacuum ports, for removing excess liquid from the chamber. In some embodiments, the vacuum port in the lower plate may be controlled after the chamber is substantially filled with additional fluids and / or reagents.

[0034] Another aspect of the present disclosure is a system comprising one or more independently operable sample processing assemblies, one or more dispensing devices, one or more optional liquid removal devices, one or more optional mixing devices, and a control system communicatively coupled to the one or more sample processing assemblies and at least the one or more dispensing devices. In some embodiments, each of the one or more independently operable sample processing assemblies comprises one or more lower plates and one or more upper plates, wherein each of the one or more upper plates is complementary to each of the one or more lower plates.

[0035] In some embodiments, the lower plate has a modular design. In some embodiments, the modular lower plate includes a main body with a lower engagement surface. In some embodiments, the modular lower plate includes a thermal management module. In some embodiments, the main body can rest on, engage with, or be coupled to the thermal management module. In some embodiments, the main body includes a substrate stage that is integral to and integral with the main body. In other embodiments, the main body includes the lower engagement surface but does not include an integral substrate stage. Conversely, the modular lower plate includes a main body with a detachable substrate stage. In this manner, the detachable substrate stage can serve as a carrier for substrates. For example, a substrate can be placed on the detachable substrate stage, and the substrate and detachable substrate stage pair can be picked up, transported, and deposited onto the main body together. In some embodiments, the substrate and the separate substrate stage pair remain together during all or at least some processing steps, for example, during all or part of the steps used to prepare a sample for microscopic analysis, such as from baking to coverslipping or from baking to staining. In some embodiments, the user places the substrate carrying the sample on the separate substrate stage and enters the substrate / substrate stage pair into the system. After sample processing, the substrate / substrate stage is retrieved from the system and the substrate with the processed sample is then removed for analysis.

[0036] In some embodiments, the system includes at least two independently operable sample processing assemblies, and wherein one of the at least two sample processing assemblies includes a sealed chamber (e.g., a chamber formed by an upper plate and a complementary lower plate or complementary modular lower plates).

[0037] In some embodiments, the lower plate and the upper plate include one or more independently operable heating and / or cooling elements. For example, independently operable heating and / or cooling elements may be present in the lower plate, the upper plate, or both. In some embodiments, the chamber is in communication with at least two heating and / or cooling elements. In some embodiments, the heating and / or cooling elements in the upper plate may operate in conjunction with those in the lower plate. By independently controlling the various heating and / or cooling elements present in the upper and lower plates, the temperature of the substrate table, any portion of the substrate supported by the substrate table, and / or the chamber may be controlled.

[0038] In some embodiments, the at least two heating and / or cooling elements can establish a thermal gradient between different portions of the lower plate and / or upper plate. In some embodiments, the thermal gradient established enables a sample and / or a portion of the substrate disposed on the substrate to be maintained at a temperature lower than the temperature of any other component in the chamber during a demasking operation. For example, the various heating and / or cooling elements present in the lower plate and / or upper plate can be independently operated so that at least a portion of the substrate or the sample disposed on the substrate remains the coldest component in the chamber during a demasking operation. For example, the substrate or the sample disposed on the substrate can be maintained at a temperature lower than the upper plate, the lower plate, the port, the valve, and / or any other structure in the chamber formed by the upper and lower plates during a demasking operation.

[0039] In some embodiments, the duration of the demasking operation is standardized for all sample types and all types of analyses to be performed on a given sample type, thereby improving the efficiency of the system and all components therein. For example, having a standardized duration for all sample and analysis types allows for easier scheduling of preceding sample processing steps (such as deparaffinization) and subsequent sample processing steps (such as staining steps), as they can all be "locked in step" with the standardized demasking operation duration. Standardization of the duration of the demasking operation is made possible by the observation that only the temperature needs to be changed to alter the degree of demasking for a given sample type. By selecting a specific temperature for performing the demasking operation for a particular sample type, not only can the duration of the demasking operation be standardized, but the degree of demasking for a particular sample type can also be optimized for a particular assay. In some embodiments, the disclosed system can be optimized to facilitate standardization of the duration of the demasking operation. Thus, in some embodiments, the demasking operation performed in each independently operable chamber is performed for the same duration, regardless of whether the biomarker is demasked in any individual chamber.

[0040] Another aspect of the present disclosure is a system comprising one or more independently operable sample processing components, wherein the one or more independently operable sample processing components are adapted to independently process and / or move a sample-carrying substrate from one processing area to another while maintaining the sample-carrying substrate in a horizontal position during all steps of the processing (e.g., during treatment with one or more fluids and / or reagents; and during demasking operations). In some embodiments, the horizontal processing (and / or horizontal movement) enables the one or more fluids and / or reagents dispensed on the sample-carrying substrate to be maintained and / or manipulated on the surface of the substrate. In some embodiments, the one or more sample processing components comprise a chamber configured to process the sample-carrying substrate at an elevated temperature and / or pressure (compared to the temperature and / or pressure outside the chamber) while maintaining the substrate in a horizontal position and while minimizing evaporation losses. In some embodiments, the chamber is formed by a lower plate having a lower engaging surface and an upper plate having an upper engaging surface, wherein the upper engaging surface is complementary to the lower engaging surface.

[0041] In some embodiments, the chamber is formed by a modular lower plate. In some embodiments, the modular lower plate includes a main body having a lower engagement surface. In some embodiments, the modular lower plate includes a thermal management module. In some embodiments, the main body can rest on, engage with, or be coupled to the thermal management module. In some embodiments, the main body includes a substrate stage that is integral with and integral to the main body. In other embodiments, the main body includes the lower engagement surface but does not include an integral substrate stage. Conversely, the modular lower plate includes a main body having a detachable substrate stage. In this manner, the detachable substrate stage can be used as a carrier for a substrate. For example, a substrate can be disposed on the detachable substrate stage, and the substrate and detachable substrate stage pair can be picked up, transported, and deposited onto the main body together. In some embodiments, the substrate and the separate substrate stage pair remain together during all or at least some processing steps, for example, during all or part of the steps used to prepare a sample for microscopic analysis, such as from baking to coverslipping or from baking to staining. In some embodiments, the user places a substrate bearing a sample on a separate substrate stage and imports the substrate / substrate stage pair into the system and after sample processing, retrieves the substrate / substrate stage from the system and then removes the substrate with the processed sample thereon for analysis.

[0042] In some embodiments, the lower plate and the upper plate move independently together such that a sealing engagement is formed between the complementary lower and upper engagement surfaces. In some embodiments, the formed chamber is configured to perform one or more steps of a demasking operation, such as any steps of heating, pressurizing, cooling, depressurizing, quenching, and / or adding or removing fluids and / or reagents.

[0043] In some embodiments, one or more fluids and / or reagents are dispensed into a sample disposed on the substrate, a portion of the substrate itself, and / or a reservoir within the lower plate prior to forming the sealed chamber. For example, a total volume of between approximately 250 μL and approximately 1000 μL of one or more fluids and / or reagents is dispensed into a sample disposed on the substrate, a portion of the substrate itself, and / or a reservoir within the lower plate prior to forming the sealed chamber. In some embodiments, the demasking operation is performed using the fluids and / or reagents supplied to the substrate prior to forming the chamber. For example, no additional fluids and / or reagents are dispensed into the substrate while it is positioned within the chamber and during the demasking operation.

[0044] In some embodiments, the chamber is in communication with one or more independently operable heating and / or cooling elements that enable the substrate, sample, and / or one or more fluids and / or reagents disposed within the chamber to be selectively heated and / or cooled to a predetermined temperature. In some embodiments, a heating fluid (such as a fluid contained in a separate reservoir on the substrate or within the chamber) enables the chamber to be heated and / or pressurized. For example, the heating fluid can enable demasking operations within a sealed chamber and on a sample disposed on a substrate. In some embodiments, the heating and / or pressurization of the chamber is monitored by one or more temperature and / or pressure sensors disposed within the chamber or in communication with at least one of the sample or substrate. In some embodiments, heating is achieved by a conductive heating element positioned on or within the substrate stage, a conductive heating element within the body of one or both of the upper and lower plates, other types of heating devices positioned adjacent to the heated fluids and / or reagents, microwave heating of the reagents using the reaction chamber, and / or magnetic induction. In some embodiments, during the demasking operation, at least a portion of the sample disposed on the substrate and / or at least a portion of the substrate itself is maintained at a lower temperature than any other components within the chamber. For example, during a demasking operation, a sample disposed on a substrate or the substrate itself may remain the "coldest" component within the chamber.

[0045] In some embodiments, the sealed chamber is heated to a predetermined temperature and / or pressurized to a predetermined pressure for a predetermined amount of time. For example, the sealed chamber may be heated at a temperature ranging from about 115° C. to about 155° C. and a pressure ranging from about 150 kPa to about 1050 kPa for a period of time ranging from about 2 minutes to about 10 minutes. In some embodiments, a portion of the sample and / or substrate remains the "coldest" component within the chamber throughout the heating and / or pressurization process.

[0046] In some embodiments, the demasking operation includes a temperature ramp-up phase, a temperature holding phase, and a temperature ramp-down phase. In some embodiments, during the temperature ramp-up phase, the temperature holding phase, and the temperature ramp-down phase, the substrate or a portion of the substrate itself disposed on the substrate is maintained as the "coldest" component in the chamber. In some embodiments, the demasking operation does not include a temperature ramp-down phase. For example, the sealed chamber can be opened while heating and / or pressurizing. In some embodiments, before opening the heating and / or pressurizing chamber, some pressure is released through one or more valves in communication with the chamber.

[0047] In some embodiments, additional fluids and / or reagents are added to the sample and / or substrate during one or more of the temperature ramp-up phase, the temperature hold phase, and the temperature ramp-down phase. For example, after the chamber is formed, between approximately 250 μL and approximately 1000 μL of one or more additional fluids and / or reagents are dispensed into a portion of the sample or substrate itself. In some embodiments, the temperature of the one or more additional fluids and / or reagents dispensed into the sealed chamber is lower than the temperature inside the heated and / or pressurized chamber. For example, the temperature of the one or more additional fluids and / or reagents can be at least 25°C lower than the temperature of the sample or the temperature inside the sealed chamber. In some embodiments, the entire sealed chamber is substantially filled with the one or more liquids. For example, if the total chamber volume is approximately 2 mL (not accounting for the volume of the substrate containing the chamber), approximately 2 mL of additional fluids and / or reagents can be added while the chamber is sealed. In some embodiments, between approximately 250 μL and approximately 1000 μL of one or more additional fluids and / or reagents are dispensed into a reservoir within the chamber, such as a heated fluid reservoir. In alternative embodiments, the fluid reservoir is maintained as the coolest component in the chamber.

[0048] In certain embodiments, the chamber is communicated with one or more ports, which facilitate delivery of one or more gases and / or steam to the chamber so that the chamber is pressurized and / or heated substrate, sample and / or one or more fluids and / or reagents disposed therein. In certain embodiments, the lower plate may comprise one or more ports, such as a vacuum port, for removing excess liquid from the chamber. In certain embodiments, after substantially filling the chamber with additional fluid and / or reagent, the vacuum port in the lower plate may be activated.

[0049] Another aspect of the present disclosure is a system comprising (a) one or more independently operable sample processing components, wherein the one or more independently operable sample processing components comprise (i) a lower plate, wherein the lower plate comprises a lower engagement surface and one or more substrate stages elevated relative to the lower engagement surface, and wherein the one or more elevated substrate stages comprise an upper surface adapted to horizontally hold a substrate; and (ii) an upper plate having an upper engagement surface complementary to the lower engagement surface, and wherein the upper plate further comprises one or more cavities recessed relative to the upper engagement surface, wherein the one or more recessed cavities are adapted to receive at least a portion of the elevated substrate stages; (b) one or more dispensing devices; and (c) a control system communicatively coupled to at least the one or more independently operable sample processing components and / or the one or more dispensing devices.

[0050] In some embodiments, at least one of the lower plate and the upper plate includes one or more independently operable heating and / or cooling elements. For example, the lower plate may include one, two, or three independently operable heating and / or cooling elements; and the upper plate may include one heating and / or cooling element. In some embodiments, at least one heating and / or cooling element is embedded within the body of the lower plate; and at least one heating and / or cooling element is embedded within the body of the upper plate. In some embodiments, a control system communicates with the one or more independently operable heating and / or cooling elements. In some embodiments, any heating and / or cooling element within the upper plate can be operated in conjunction with those within the lower plate. By independently controlling the various heating and / or cooling elements present in the upper and lower plates, the temperature of the substrate table, any portion of the substrate supported by the substrate table, and / or the chamber can be controlled. For example, the various heating and / or cooling elements present in the lower and / or upper plates can be independently operated so that at least a portion of the substrate or a sample disposed on the substrate is maintained as the coldest structure within the chamber. For example, a substrate or a sample disposed on the substrate can be maintained at a lower temperature than the upper plate, the lower plate, ports, valves, and / or any other structure within the chamber formed by the upper and lower plates. In some embodiments, the heating and / or cooling elements are each independently operated to maintain a predetermined temperature gradient between different portions of the upper and lower plates and the substrate stage.

[0051] In some embodiments, the one or more independently operable heating and / or cooling elements are in thermal communication with the one or more substrate tables. In some embodiments, the independently operable heating and / or cooling elements are configured such that a sample disposed on a substrate and positioned on a surface of the substrate table has a lower temperature than any other component in thermal communication with the one or more independently operable heating and / or cooling elements.

[0052] In some embodiments, each of the one or more dispensing devices comprises one or more dispensing nozzles, one or more pipettes, and / or one or more on-demand dispensing devices. For example, the dispensing device can be commanded by a control system to dispense one or more fluids and / or reagents in a total volume ranging from about 250 μL to about 1000 μL. In some embodiments, the system further comprises one or more mixing devices and / or one or more liquid removal devices. In some embodiments, the one or more dispensing devices are coupled to a dispensing subassembly. In some embodiments, the system comprises a plurality of sample processing assemblies. In some embodiments, at least one of the plurality of sample processing assemblies comprises a chamber formed by an upper plate and a lower plate. In some embodiments, the base is horizontally disposed within the chamber.

[0053] Another aspect of the present disclosure is a system comprising: (a) a plurality of independently operable sample processing assemblies, wherein each independently operable sample processing assembly comprises (i) a lower plate movably coupled to a lower track, wherein the lower plate comprises a lower engagement surface and one or more substrate tables elevated relative to the lower engagement surface, and wherein the one or more elevated substrate tables comprise an upper surface adapted to hold a substrate horizontally as the lower plate traverses the lower track; and (ii) an upper plate having an upper engagement surface complementary to the lower engagement surface, and wherein the upper plate further comprises one or more cavities recessed relative to the upper engagement surface, wherein the one or more recessed cavities are adapted to receive at least a portion of the one or more elevated substrate tables; and wherein the lower plate is configured such that the flat upper surface of the substrate table remains horizontal as the lower plate traverses the lower track; (b) one or more dispensing devices; and (c) a control system in communication with the plurality of independently operable sample processing assemblies and / or the one or more dispensing devices.

[0054] In some embodiments, a control system is adapted to maintain the environment within a chamber formed by the complementary lower and upper plates, for example, by controlling one or more heating elements, one or more cooling elements, and / or one or more gases and / or a steam generation and delivery unit in communication with the chamber. In some embodiments, the one or more heating and / or cooling elements are independently controlled so that a sample disposed on the substrate and / or a portion of the substrate itself is maintained at a temperature lower than any other components (e.g., ports, valves, sensors, probes, etc.) within the formed chamber. In some embodiments, the control system monitors the temperature and / or pressure within the chamber (e.g., using one or more temperature and / or pressure sensors within the chamber and / or in contact with the substrate) and increases and / or decreases the temperature and / or pressure to maintain a predetermined temperature and / or predetermined pressure. For example, the temperature within the chamber can be monitored using a temperature sensor communicatively coupled to the control system. When the temperature within the chamber reaches a predetermined threshold chamber temperature, the control system can command the one or more heating elements to maintain a steady state or to shut down.

[0055] In some embodiments, the control system commands one or more force-generating members to apply a predetermined external force to at least one of the upper plate or the lower plate to maintain the upper and lower engaging surfaces of the upper and lower plates, respectively, in sealing engagement, particularly when pressure within any formed chamber increases. In some embodiments, the control system commands any force-generating member to apply a predetermined amount of force that is less than the force generated at a predetermined threshold pressure. In some embodiments, the control system can command one or more ports and / or valves to release pressure above the predetermined threshold pressure.

[0056] In some embodiments, the one or more substrate tables are configured such that any substrate supported by the one or more substrate tables is maintained in a horizontal position, and remains in a horizontal position during movement of the lower plate within the system, regardless of whether the movement is entirely horizontal or whether the movement includes a vertical component (or even an entirely vertical movement).

[0057] In some embodiments, at least one of the sample processing components includes a sealed chamber. In some embodiments, the system further includes a substrate at least partially disposed within the sealed chamber, wherein the substrate is supported by the one or more substrate stages and oriented in a horizontal position. In some embodiments, the sample disposed on the substrate is treated with one or more fluids and / or reagents outside the chamber while in the horizontal position, and wherein the demasking operation is performed within the chamber while the substrate is disposed in the horizontal position. In some embodiments, no additional fluids and / or reagents are dispensed to the substrate while the substrate is positioned within the chamber. In some embodiments, the demasking operation is performed using only the fluids and / or reagents dispensed to the substrate prior to forming the chamber. In some embodiments, during all stages of the demasking operation, the sample disposed on the substrate, or the substrate itself, is maintained as the "coldest" component within the chamber. For example, during the temperature ramp-up phase, the temperature hold phase, and the temperature ramp-down phase of the demasking operation, the substrate and / or a portion of the substrate is maintained as the "coldest" component within the chamber.

[0058] In some embodiments, the lower track of each of the plurality of independently operable sample processing assemblies is horizontally positioned, e.g., each lower track is arranged parallel to the ground. In some embodiments, the lower track of each of the plurality of independently operable sample processing assemblies includes a first end that is vertically elevated relative to a second end, such that each lower track is offset from horizontal, e.g., the track is offset relative to horizontal.

[0059] In some embodiments, the system further includes a motor adapted to move the lower plate from a first position along the lower track to a second position along the lower track. In some embodiments, the second position along the lower track is below the upper plate, such as at a predetermined position below a pre-positioned upper plate. In some embodiments, the second position along the first lower track is in a preparation area, and wherein the upper plate is moved (in any one of an x-direction, a y-direction, and a z-direction) to the pre-positioned lower plate.

[0060] In some embodiments, the dispensing device is coupled to a dispensing track, wherein the dispensing track is oriented perpendicular to a lower track of each of the plurality of independently operable sample processing assemblies (regardless of whether the lower track is arranged horizontally or offset relative to the horizontal). In some embodiments, the upper plate is coupled to a force generating member. In some embodiments, the force generating member is selected from the group consisting of a lever, a screw, a motor, a spring, a pneumatic piston, a hydraulic piston, a cam mechanism, and any combination thereof. In some embodiments, the upper plate is secured to the subassembly.

[0061] In some embodiments, the disclosed system includes at least two independently operable sample processing components. In some embodiments, one of the at least two independently operable sample processing components includes a chamber.

[0062] Another aspect of the present disclosure is a sample processing assembly comprising: (i) a lower plate comprising: (a) a substrate table having an upper surface adapted to horizontally support a substrate; (b) a lower engaging surface at least partially surrounding the substrate table; and (c) a first lower temperature adjustment element in thermal communication with the substrate table; and (ii) an upper plate comprising: (a) an upper engaging surface complementary to the lower engaging surface; and (b) a cavity. In some embodiments, the first lower temperature adjustment element is positioned below the substrate table. In some embodiments, the lower plate further comprises a second lower temperature adjustment element and a third lower temperature adjustment element. In some embodiments, each temperature adjustment element is independently operable. In some embodiments, the second lower temperature adjustment element and the third lower temperature adjustment element are each positioned adjacent to the first lower temperature adjustment element. In some embodiments, the second lower temperature adjustment element and the third lower temperature adjustment element are each positioned below a portion of the lower engaging surface. In some embodiments, the first lower engaging element, the second lower engaging element, and the third lower engaging element are positioned parallel to one another.

[0063] In some embodiments, a thermal gradient is maintained between the first and second lower temperature-regulating elements and between the first and third temperature-regulating elements. In some embodiments, the thermal gradient maintained between the first and second lower temperature-regulating elements is in a range of 2°C to about 10°C; and wherein the thermal gradient maintained between the first and second lower temperature-regulating elements is in a range of 2°C to about 10°C. In some embodiments, the thermal gradient maintained between the first and second lower temperature-regulating elements is in a range of 2°C to about 5°C; and wherein the thermal gradient maintained between the first and second lower temperature-regulating elements is in a range of 2°C to about 5°C. In some embodiments, the first, second, and third lower temperature-regulating elements are positioned to maintain a thermal gradient between at least a portion of the lower bonding surface and the substrate table.

[0064] In some embodiments, the first lower temperature-regulating element has a first thermal output, the second lower temperature-regulating element has a second thermal output, and the third lower temperature-regulating element has a third thermal output, wherein the first thermal output is less than either the second thermal output or the third thermal output. In some embodiments, the upper plate further comprises at least one upper thermal-regulating element. In some embodiments, the thermal output of the first lower temperature-regulating element is maintained at a temperature lower than the thermal output of the at least one upper thermal-regulating element.

[0065] In some embodiments, the sample processing assembly further includes a substrate disposed on a surface of the substrate table. In some embodiments, the substrate is maintained at a temperature lower than the temperature of the lower engagement surface and the upper engagement surface. In some embodiments, the sample or portion of the substrate disposed on the substrate is maintained at a temperature lower than any other component within the chamber, e.g., during a demasking operation, the sample or portion of the substrate disposed on the substrate is the "coldest" component within the chamber.

[0066] In some embodiments, the first lower temperature adjustment element includes at least one fluid channel. In some embodiments, the second lower temperature adjustment element and the third lower temperature adjustment element each include a heating cartridge. In some embodiments, at least one of the lower plate and the upper plate includes at least one sealing body. In some embodiments, the at least one sealing body is removable. In some embodiments, at least one of the upper plate or the lower plate is coupled to at least one of a motor, a piston, a spring, a screw mechanism, a lever, or a cam mechanism.

[0067] In some embodiments, the sample processing assembly further includes a subassembly having a first end and a second end. In some embodiments, the lower plate is movable along the length of the subassembly between the first end and the second end.

[0068] In some embodiments, the subassembly is arranged horizontally. In some embodiments, the lower plate and the upper plate are independently moved to a predetermined position such that the lower engaging surface of the lower plate at least partially contacts the complementary upper engaging surface of the upper plate. In some embodiments, the lower plate is moved to the predetermined position, and then the upper plate is moved toward the lower plate. In some embodiments, the movement toward the lower plate is performed using one of a motor, a piston, or a cam mechanism.

[0069] In some embodiments, the subassembly is offset from a horizontal orientation. In some embodiments, the upper plate remains stationary and wherein the lower plate moves toward the upper plate until a lower engagement surface of the lower plate at least partially contacts a complementary upper engagement surface of the upper plate. In some embodiments, the upper plate and the lower plate have complementary wedge-like shapes. In some embodiments, the lower plate and the upper plate both independently move to a predetermined position such that the lower engagement surface of the lower plate at least partially contacts a complementary upper engagement surface of the upper plate. In some embodiments, the lower plate and the upper plate are both independently movable.

[0070] Another aspect of the present disclosure is a method for demasking a sample disposed on a substrate, the method comprising: (a) dispensing a predetermined volume of one or more fluids and / or reagents onto at least a portion of the sample; (b) sealing the sample within a chamber, wherein the chamber is formed by contacting (i) a lower engaging surface of a lower plate with (ii) an upper engaging surface of an upper plate, wherein the upper engaging surface is complementary to the lower engaging surface, and wherein the lower plate further comprises a substrate stage for supporting the substrate; and (c) performing a demasking operation on the sample within the sealed chamber. In some embodiments, the lower plate includes one or more independently operable heating and / or cooling elements. In some embodiments, the upper plate includes one or more independently operable heating and / or cooling elements. In some embodiments, both the lower and upper plates include operable heating and / or cooling elements. In some embodiments, any heating and / or cooling elements within the upper plate can operate in conjunction with those within the lower plate. By independently controlling the various heating and / or cooling elements present in the upper and lower plates, the temperature of the substrate stage, any portion of the substrate supported by the substrate stage, and / or the chamber can be controlled.

[0071] In some embodiments, the chamber is formed by a lower plate having a modular design. In some embodiments, the modular lower plate includes a main body having a lower engagement surface. In some embodiments, the modular lower plate includes a thermal management module. In some embodiments, the main body can rest on, engage with, or be coupled to the thermal management module. In some embodiments, the main body includes a substrate stage that is integral with and integral to the main body. In other embodiments, the main body includes the lower engagement surface but does not include an integral substrate stage. Conversely, the modular lower plate includes a main body having a detachable substrate stage. In this manner, the detachable substrate stage can be used as a carrier for a substrate. For example, a substrate can be placed on the detachable substrate stage, and the substrate and detachable substrate stage pair can be picked up, transported, and deposited onto the main body together. In some embodiments, the substrate and the separate substrate stage pair remain together during all or at least some processing steps, for example, during all or part of the steps used to prepare a sample for microscopic analysis, such as from baking to coverslipping or from baking to staining. In some embodiments, the user places a substrate bearing a sample on a separate substrate stage and imports the substrate / substrate stage pair into the system and after sample processing, retrieves the substrate / substrate stage from the system and then removes the substrate with the processed sample thereon for analysis.

[0072] In some embodiments, the one or more fluids and / or reagents are selected from water and a buffer solution having a pH range of about 5 to about 10. In some embodiments, the one or more fluids and / or reagents comprise a mixture of deionized water, tris(hydroxymethyl)methylamine, and a chelating agent. In some embodiments, the predetermined volume of the one or more fluids and / or reagents dispensed onto the at least a portion of the sample is in a range of about 200 μL to about 1000 μL. In some embodiments, the predetermined volume of the one or more fluids and / or reagents dispensed onto the at least a portion of the sample is in a range of about 250 μL to about 500 μL.

[0073] In some embodiments, the demasking operation includes heating the sample disposed on the substrate to a first predetermined temperature for a predetermined duration. In some embodiments, the first predetermined temperature is in a range of approximately 125°C to approximately 155°C. In some embodiments, the first predetermined temperature is in a range of approximately 135°C to approximately 150°C. In some embodiments, the first predetermined temperature is approximately 140°C. In some embodiments, the predetermined duration is in a range of approximately 1 minute to approximately 10 minutes. In some embodiments, the predetermined duration is in a range of approximately 1 minute to approximately 7 minutes. In some embodiments, the predetermined duration is in a range of approximately 1 minute to approximately 5 minutes.

[0074] In some embodiments, during the demasking operation, the temperature of the substrate is lower than the temperature of any other components in the chamber. In some embodiments, the temperature of the substrate is at least 10° C. lower than the temperature of other components in the chamber. In some embodiments, the temperature of the substrate is at least 5° C. lower than the temperature of other components in the chamber.

[0075] In some embodiments, less than about 5% of the predetermined volume of the one or more fluids and / or reagents allocated to the sample is lost due to evaporation during the unmasking operation. In some embodiments, less than about 2% of the predetermined volume of the one or more fluids and / or reagents allocated to the sample is lost due to evaporation during the unmasking operation. In some embodiments, after the chamber is sealed, substantially no further fluids and / or reagents are allocated to the substrate. In some embodiments, after the chamber is sealed, no further fluids and / or reagents are allocated to the substrate.

[0076] In some embodiments, the method further comprises pre-pressurizing the sealed chamber. In some embodiments, the method further comprises introducing steam into the sealed chamber.

[0077] In some embodiments, the lower plate further comprises at least one heating element. In some embodiments, the lower plate further comprises at least three heating elements, wherein a first of the at least three heating elements is positioned below the substrate table, and wherein a second and a third of the at least three heating elements are respectively positioned adjacent to the first of the at least three heating elements. In some embodiments, during the demasking operation, a first thermal gradient is maintained between the first and second of the at least three heating elements, and wherein during the demasking operation, a second thermal gradient is maintained between the second and third of the at least three heating elements.

[0078] In some embodiments, a force generating member applies an external force to the sealed chamber. In some embodiments, the force generating member is selected from the group consisting of a motor, a piston, a spring, a screw mechanism, a lever, and a cam mechanism. In some embodiments, at least one of the lower plate and the upper plate is thermally connected to a thermal management module. In some embodiments, the demasking operation includes a temperature ramp phase and a temperature hold phase. In some embodiments, the temperature ramp phase includes heating the sample at a rate in a range of about 1°C / s to about 4°C / s. In some embodiments, the temperature ramp phase includes heating the sample at a rate in a range of about 3°C / s to about 4°C / s.

[0079] In some embodiments, the demasking operation further includes a temperature ramp-down phase. In some embodiments, the temperature ramp-up phase is shorter than the temperature ramp-down phase. In some embodiments, the temperature ramp-down phase includes cooling the sample at a rate in a range of about 0.5°C / s to about 3°C / s.

[0080] In some embodiments, the demasking operation is stopped by opening the chamber without a temperature ramp-down phase. In some embodiments, the demasking operation is stopped by introducing a predetermined amount of fluid into the chamber. In some embodiments, the predetermined amount of fluid introduced to stop the demasking operation is in a range of about 0.5 mL to about 5 mL.

[0081] In some embodiments, the method further comprises staining the sample for the presence of one or more biomarkers after the unmasking operation is completed.

[0082] Another aspect of the present disclosure is a method for demasking a sample disposed on a substrate, the method comprising: (a) dispensing a predetermined volume of one or more fluids and / or reagents onto at least a portion of the sample; (b) sealing the sample within a chamber, wherein the chamber comprises (i) a lower plate having a substrate stage and a lower engagement surface, and (ii) an upper plate having an upper engagement surface complementary to the lower engagement surface, and wherein the substrate is supported in a horizontal position by the substrate stage; and (c) performing a demasking operation within the sealed chamber. In some embodiments, the one or more fluids and / or reagents dispensed to the sample are selected from the group consisting of water and a buffer solution. In some embodiments, the predetermined volume of the one or more fluids and / or reagents dispensed to the sample is in a range from about 200 μL to about 1000 μL. In some embodiments, the predetermined volume of the one or more fluids and / or reagents dispensed to the sample is in a range from about 250 μL to about 500 μL.

[0083] In some embodiments, the lower plate includes one or more independently operable heating and / or cooling elements. In some embodiments, the upper plate includes one or more independently operable heating and / or cooling elements. In some embodiments, both the lower and upper plates include independently operable heating and / or cooling elements. In some embodiments, any heating and / or cooling elements in the upper plate can operate in conjunction with those in the lower plate. By independently controlling the various heating and / or cooling elements present in the upper and lower plates, the temperature of the substrate table, any portion of the substrate supported by the substrate table, and / or the chamber can be controlled.

[0084] In some embodiments, the lower plate has a modular design. In some embodiments, the modular lower plate includes a main body with a lower engagement surface. In some embodiments, the modular lower plate includes a thermal management module. In some embodiments, the main body can rest on, engage with, or be coupled to the thermal management module. In some embodiments, the main body includes a substrate stage that is integral to and integral with the main body. In other embodiments, the main body includes the lower engagement surface but does not include an integral substrate stage. Conversely, the modular lower plate includes a main body with a detachable substrate stage. In this manner, the detachable substrate stage can serve as a carrier for substrates. For example, a substrate can be placed on the detachable substrate stage, and the substrate and detachable substrate stage pair can be picked up, transported, and deposited onto the main body together. In some embodiments, the substrate and the separate substrate stage pair remain together during all or at least some processing steps, for example, during all or part of the steps used to prepare a sample for microscopic analysis, such as from baking to coverslipping or from baking to staining. In some embodiments, the user places the substrate carrying the sample on the separate substrate stage and enters the substrate / substrate stage pair into the system. After sample processing, the substrate / substrate stage is retrieved from the system and the substrate with the processed sample is then removed for analysis.

[0085] In some embodiments, the demasking operation includes heating the sample disposed on the substrate to a first predetermined temperature for a predetermined duration. In some embodiments, the first predetermined temperature is in the range of about 125°C to about 155°C. In some embodiments, the predetermined duration is in the range of about 1 minute to about 10 minutes. In some embodiments, during the demasking operation, the temperature of the substrate is lower than the temperature of any other components within the chamber. In some embodiments, the temperature of the substrate is at least 5°C lower than the temperature of the other components within the chamber. In some embodiments, less than about 5% of the predetermined volume of the one or more fluids and / or reagents dispensed to the sample is lost due to evaporation during the demasking operation. In some embodiments, after the chamber is sealed, no further fluids and / or reagents are dispensed to the substrate.

[0086] In some embodiments, the method further comprises pre-pressurizing the sealed chamber prior to performing the demasking operation. In some embodiments, the chamber is pre-pressurized while performing the demasking operation. In some embodiments, the method further comprises introducing steam into the sealed chamber.

[0087] In some embodiments, the demasking operation includes heating the sample at a rate in a range of about 1°C / s to about 4°C / s. In some embodiments, the demasking operation includes heating the sample at a rate in a range of about 3°C / s to about 4°C / s. In some embodiments, the demasking operation includes cooling the sample at a rate in a range of about 0.5°C / s to about 2.5°C / s.

[0088] In another aspect of the present disclosure, a method for demasking a sample disposed on a substrate comprises: (a) dispensing a predetermined volume of one or more fluids and / or reagents onto at least a portion of the sample; (b) sealing the sample within a chamber, wherein the chamber comprises a lower plate having a substrate table and a lower engagement surface, and an upper plate having an upper engagement surface complementary to the lower engagement surface, and wherein the substrate is supported in a horizontal position by the substrate table; and (c) performing a demasking operation, wherein the demasking operation comprises a temperature ramp-up phase, a temperature hold phase, and a temperature ramp-down phase. In some embodiments, the temperature ramp-up phase comprises heating the sample at a rate within a range of about 1°C / s to about 4°C / s. In some embodiments, the temperature ramp-up phase comprises heating the sample at a rate within a range of about 3°C / s to about 4°C / s. In some embodiments, the temperature ramp-up phase comprises heating the sample to a predetermined temperature within a range of about 110°C to about 150°C. In some embodiments, the temperature ramp-up phase comprises heating the sample to a predetermined temperature within a range of about 120°C to about 145°C.

[0089] In some embodiments, the sample is maintained at the predetermined temperature for a predetermined period of time in a range of about 1 minute to about 10 minutes. In some embodiments, the sample is maintained at the predetermined temperature for a predetermined period of time in a range of about 2 minutes to about 7 minutes. In some embodiments, the sample is maintained at the predetermined temperature for a predetermined period of time in a range of about 3 minutes to about 5 minutes.

[0090] In some embodiments, the temperature ramp down phase comprises cooling the sample at a rate in a range between about 0.5°C / s and about 3°C / s. In some embodiments, the temperature ramp down phase comprises cooling the sample at a rate in a range between about 0.5°C / s and about 2°C / s.

[0091] In some embodiments, the one or more fluids and / or reagents are selected from the group consisting of water and a buffer solution. In some embodiments, the predetermined volume of the one or more fluids and / or reagents dispensed into the sample is in a range of about 200 μL to about 1000 μL. In some embodiments, the predetermined volume of the one or more fluids and / or reagents dispensed into the sample is in a range of about 250 μL to about 500 μL.

[0092] Another aspect of the present disclosure is an antigen retrieval sample prepared by: (a) dispensing a predetermined volume of one or more fluids and / or reagents to at least a portion of the sample; (b) sealing the sample within a chamber, wherein the chamber includes a lower plate having a substrate stage and a lower engagement surface, and an upper plate having an upper engagement surface complementary to the lower engagement surface, and wherein the substrate is supported in a horizontal position by the substrate stage; and (c) performing a demasking operation within the sealed chamber.

[0093] Another aspect of the present disclosure is a target repair sample prepared by: (a) dispensing a predetermined volume of one or more fluids and / or reagents into at least a portion of the sample; (b) sealing the sample within a chamber, wherein the chamber includes a lower plate having a substrate table and a lower engagement surface, and an upper plate having an upper engagement surface complementary to the lower engagement surface, and wherein the substrate is supported in a horizontal position by the substrate table; and (c) performing a demasking operation within the sealed chamber.

[0094] Another aspect of the present disclosure is a target retrieval sample prepared by: (a) dispensing a predetermined volume of one or more fluids and / or reagents into a portion of the sample; (b) sealing the sample within a chamber, wherein the chamber is formed by contacting a lower engagement surface of a lower plate with an upper engagement surface of an upper plate, wherein the upper engagement surface is complementary to the lower engagement surface, and wherein the lower plate further comprises a substrate stage for supporting a substrate; and (c) performing a demasking operation on the sample within the sealed chamber. In some embodiments, the antigen retrieval sample is a biopsy sample.

[0095] Another aspect of the present disclosure is an antigen retrieval sample prepared by: (a) dispensing a predetermined volume of one or more fluids and / or reagents to at least a portion of the sample; (b) sealing the sample within a chamber, wherein the chamber comprises a lower plate having a substrate stage and a lower engagement surface and an upper plate having an upper engagement surface complementary to the lower engagement surface, and wherein the substrate is supported in a horizontal position by the substrate stage; and (c) performing a demasking operation, wherein the demasking operation comprises a temperature ramp-up phase, a temperature hold phase, and a temperature ramp-down phase. In some embodiments, during all phases of the demasking operation, e.g., during the temperature ramp-up phase, during the temperature hold phase, and during the temperature ramp-down phase, the sample is retained as the "coldest" component within the sealed chamber.

[0096] Another aspect of the present disclosure is a target repair sample prepared by: (a) dispensing a predetermined volume of one or more fluids and / or reagents to at least a portion of the sample; (b) sealing the sample within a chamber, wherein the chamber includes a lower plate having a substrate table and a lower engagement surface and an upper plate having an upper engagement surface complementary to the lower engagement surface, and wherein the substrate is supported in a horizontal position by the substrate table; and (c) performing a demasking operation, wherein the demasking operation includes a temperature ramp-up phase, a temperature hold phase, and a temperature ramp-down phase. In some embodiments, during all phases of the demasking operation, e.g., during the temperature ramp-up phase, during the temperature hold phase, and during the temperature ramp-down phase, the sample is retained as the "coldest" component within the sealed chamber.

[0097] Another aspect of the present disclosure is an unmasked sample disposed on a substrate, wherein the unmasked sample is prepared according to a method comprising the following steps: (a) dispensing a predetermined volume of one or more fluids and / or reagents onto a portion of the sample disposed on the substrate; (b) sealing the sample within a chamber, wherein the chamber is formed by contacting a lower engaging surface of a lower plate with an upper engaging surface of an upper plate, wherein the upper engaging surface is complementary to the lower engaging surface, and wherein the lower plate further comprises a substrate stage for supporting the substrate; and (c) performing an unmasking operation on the sample within the sealed chamber. In some embodiments, the antigen-repaired sample is a biopsy sample, and (c) performing an unmasking operation on the sample. In some embodiments, the unmasked sample comprises one or more repaired antigenic sites. In some embodiments, the unmasked sample comprises one or more repaired nucleic acid targets. In some embodiments, after sealing the chamber, no further fluids and / or reagents are dispensed to the substrate.

[0098] In some embodiments, the lower plate includes one or more independently operable heating and / or cooling elements. In some embodiments, the upper plate includes one or more independently operable heating and / or cooling elements. In some embodiments, both the lower and upper plates include independently operable heating and / or cooling elements. In some embodiments, any heating and / or cooling elements in the upper plate can operate in conjunction with those in the lower plate. By independently controlling the various heating and / or cooling elements present in the upper and lower plates, the temperature of the substrate table, any portion of the substrate supported by the substrate table, and / or the chamber can be controlled.

[0099] In some embodiments, the lower plate has a modular design. In some embodiments, the modular lower plate includes a main body having a lower engagement surface. In some embodiments, the modular lower plate includes a thermal management module. In some embodiments, the main body can rest on, engage with, or be coupled to the thermal management module. In some embodiments, the main body includes a substrate table that is integral with and part of the main body. In other embodiments, the main body includes the lower engagement surface but does not include an integral substrate table. Conversely, the modular lower plate includes a main body having a detachable substrate table. In this way, the detachable substrate table can be used as a carrier for the substrate. For example, the substrate can be placed on the detachable substrate table, and the substrate and detachable substrate table pair can be picked up, transported to, and deposited onto the main body together.

[0100] In some embodiments, the substrate and separate substrate stage pair remain together during all or at least some processing steps, for example, during all or part of the steps used to prepare a sample for microscopic analysis, such as from baking to coverslipping or from baking to staining. In some embodiments, the user places the substrate bearing the sample on a separate substrate stage and inputs the substrate / substrate stage pair into the system. After sample processing, the substrate / substrate stage is retrieved from the system, and the substrate with the processed sample thereon is then removed for analysis. In some embodiments, the one or more fluids and / or reagents are selected from the group consisting of water and a buffer solution having a pH range of about 5 to about 10. In some embodiments, the one or more fluids and / or reagents comprise a mixture of deionized water, tris(hydroxymethyl)methylamine, and a chelating agent. In some embodiments, the predetermined volume of the one or more fluids and / or reagents dispensed to the sample ranges from about 200 μL to about 1000 μL. In some embodiments, the predetermined volume of the one or more fluids and / or reagents dispensed to the sample ranges from about 250 μL to about 500 μL.

[0101] In some embodiments, the demasking operation includes heating the sample disposed on the substrate to a first predetermined temperature for a predetermined duration. In some embodiments, the first predetermined temperature is in a range of approximately 125° C. to approximately 155° C. In some embodiments, the first predetermined temperature is in a range of approximately 135° C. to approximately 150° C. In some embodiments, the first predetermined temperature is approximately 140° C. In some embodiments, the predetermined duration is in a range of approximately 1 minute to approximately 10 minutes. In some embodiments, the predetermined duration is in a range of approximately 1 minute to approximately 5 minutes.

[0102] In some embodiments, during the unmasking operation, the temperature of the substrate is lower than the temperature of any other components within the chamber. In some embodiments, the temperature of the substrate is at least 5°C lower than the temperature of other components within the chamber. In some embodiments, less than about 5% of the predetermined volume of the one or more fluids and / or reagents dispensed to the sample is lost due to evaporation during the unmasking operation. In some embodiments, less than about 2% of the predetermined volume of the one or more fluids and / or reagents dispensed to the sample is lost due to evaporation during the unmasking operation.

[0103] In some embodiments, the sealed chamber is pre-pressurized. In some embodiments, steam is further introduced into the sealed chamber.

[0104] In some embodiments, the lower plate further comprises at least one heating element. In some embodiments, the lower plate further comprises at least three heating elements, wherein a first of the at least three heating elements is positioned below the substrate table, and wherein a second and a third of the at least three heating elements are positioned adjacent to the first of the at least three heating elements. In some embodiments, during the demasking operation, a first thermal gradient is maintained between the first and the second of the at least three heating elements, and wherein during the demasking operation, a second thermal gradient is maintained between the second and the third of the at least three heating elements.

[0105] Another aspect of the present disclosure is an unmasked sample, wherein the unmasked sample is prepared in a method in which a sample disposed on a substrate is retained as the coldest component within a sealed chamber during an unmasking operation, wherein the unmasked sample is prepared by: (a) dispensing a predetermined volume of one or more fluids and / or reagents onto a portion of the sample disposed on the substrate; (b) positioning the sample-carrying substrate containing the dispensed one or more fluids and / or reagents onto a substrate stage within a chamber formed by an upper plate and a lower plate, wherein the lower plate comprises a substrate stage and a lower engagement surface, and wherein the upper plate comprises an upper engagement surface complementary to the lower engagement surface; (c) sealing the chamber; and (d) performing an unmasking operation on the sample. In some embodiments, the unmasked sample comprises one or more repaired antigenic sites. In some embodiments, the unmasked sample comprises one or more repaired nucleic acid targets. In some embodiments, after sealing the chamber, no further fluids and / or reagents are dispensed to the substrate.

[0106] Another aspect of the present disclosure relates to a system comprising: (i) at least one chamber, wherein the at least one chamber comprises an upper plate and a lower plate, wherein the lower plate comprises a lower engagement surface and one or more substrate tables adapted to hold a substrate horizontally within the at least one chamber, wherein the upper plate comprises an upper engagement surface complementary to the lower engagement surface and a cavity recessed relative to the upper engagement surface, and wherein at least one of the upper plate and the lower plate comprises at least one of a heating element or a cooling element; and (ii) a staining module. In some embodiments, the at least one chamber is a demasking chamber.

[0107] In some embodiments, at least one of the unmasking chambers has a predetermined internal volume. In some embodiments, the predetermined internal volume is approximately 14 cm 3 to about 25cm 3 In some embodiments, the predetermined internal volume is about 16 cm 3 to about 22cm 3 In some embodiments, the predetermined internal volume is about 18 cm 3 to about 20cm 3 In some embodiments, the one or more substrate tables are elevated relative to the lower engagement surface. In some embodiments, the upper plate comprises at least one steam injection port. In some embodiments, the at least one steam injection port is in fluid communication with a steam reservoir or a steam generating element. In some embodiments, the at least one chamber is configured to rapidly heat and / or pressurize a predetermined internal volume. In some embodiments, the lower plate and / or upper plate can be separated from each other after steam heating and / or pressurization.

[0108] In some embodiments, at least one of the lower plate and / or the upper plate further comprises one or more alignment members. In some embodiments, at least one of the lower plate and / or the upper plate further comprises one or more temperature sensors and / or pressure sensors. In some embodiments, at least one of the lower plate and / or the upper plate further comprises one or more temperature sensors that contact the horizontally held substrate or the fluid disposed thereon. In some embodiments, the one or more temperature sensors directly contact the horizontally held substrate or the fluid disposed thereon.

[0109] In some embodiments, the body of the lower plate and the body of the upper plate both have complementary wedge-based shapes. In some embodiments, at least one of the lower plate and the upper plate includes at least one sealing body. In some embodiments, the at least one sealing body is removable. In some embodiments, the lower plate includes a groove and wherein the at least one removable sealing body at least partially engages the groove. In some embodiments, the at least one removable sealing body is integrated into a removable sealing attachment, wherein the removable sealing attachment engages a portion of the periphery of the lower plate or the upper plate.

[0110] In some embodiments, at least one of the upper plate or the lower plate is coupled to at least one of a motor, a piston, a spring, a screw mechanism, a lever, or a cam mechanism. In some embodiments, the upper plate and the lower plate are independently movable.

[0111] In some embodiments, the system further comprises a control system. In some embodiments, the control system is adapted to operate the at least one heating element or cooling element to uniformly heat and / or cool the one or more substrate tables. In some embodiments, the control system is adapted to operate the at least one heating element or cooling element to maintain the one or more substrate tables as the coolest component within the chamber.

[0112] In some embodiments, the system includes at least one substrate transfer device. In some embodiments, the at least one substrate transfer device is selected from the group consisting of a gripper device, a forklift device, and a carrier transport device. In some embodiments, the system further includes one or more substrate loading stations.

[0113] In some embodiments, the lower plate is modular. In some embodiments, the modular lower plate is transportable to the upper plate. In some embodiments, the upper plate is coupled to the force generating member. In some embodiments, the force generating member is configured to retract if the pressure within the chamber exceeds a predetermined threshold.

[0114] Another aspect of the present disclosure is a system comprising (i) at least one demasking chamber having a predetermined interior volume, wherein the at least one demasking chamber comprises an upper plate and a lower plate, wherein the lower plate comprises a lower engagement surface and one or more substrate tables adapted to hold a substrate horizontally within the at least one demasking chamber, and wherein the upper plate comprises an upper engagement surface complementary to the lower engagement surface and a cavity recessed relative to the upper engagement surface; and wherein the upper plate comprises one or more steam injection ports for introducing steam into the at least one demasking chamber; (ii) a dyeing module; and (iii) a steam reservoir. In some embodiments, the predetermined interior volume of the at least one demasking chamber is approximately 14 cm 3 to about 25cm 3In some embodiments, the upper and lower plates can be removably coupled together (e.g., clamped together) such that upon reaching a predetermined internal pressure and / or temperature, the coupling can be disengaged and the upper and lower plates can be quickly separated from each other. In some embodiments, at least one of the lower and / or upper plates is in thermal communication with at least one heating and / or cooling element. In some embodiments, the at least one heating and / or cooling element can operate in conjunction with the one or more steam injection ports.

[0115] In some embodiments, the upper plate is coupled to a force generating member. In some embodiments, the force generating member is configured to retract if pressure within the chamber exceeds a predetermined threshold. In some embodiments, the force generating member is selected from the group consisting of a motor, a spring, a screw, a level, a piston, a cam, or any combination thereof.

[0116] In some embodiments, at least one of the lower plate and / or the upper plate further comprises one or more alignment members.

[0117] In some embodiments, at least one of the lower plate and / or the upper plate further comprises one or more temperature sensors and / or pressure sensors. In some embodiments, at least one of the lower plate and the upper plate further comprises at least one sealing body. In some embodiments, the at least one sealing body is removable. In some embodiments, the lower plate comprises a groove and wherein the at least one removable sealing body at least partially engages the groove.

[0118] Another aspect of the present disclosure is a system comprising: (i) at least one sample processing assembly comprising: (1) a lower plate comprising a body including (a) a substrate table having an upper surface adapted to horizontally support a substrate, (b) a lower engagement surface, and (c) a first lower temperature adjustment element in thermal communication with the substrate table; and (2) an upper plate comprising an upper engagement surface complementary to the lower engagement surface; and (ii) a staining module.

[0119] In some embodiments, a first lower temperature adjustment element is positioned below the substrate table. In some embodiments, the lower plate further comprises a second lower temperature adjustment element and a third lower temperature adjustment element, wherein the second lower temperature adjustment element and the third lower temperature adjustment element are each positioned adjacent to the first lower temperature adjustment element. In some embodiments, the first lower temperature adjustment element has a first heat output, the second lower temperature adjustment element has a second heat output, and the third lower temperature adjustment element has a third heat output, wherein the first heat output is less than either the second heat output or the third heat output.

[0120] In some embodiments, a thermal gradient is maintained between the first lower temperature-regulating element and the second lower temperature-regulating element and between the first temperature-regulating element and the third temperature-regulating element. In some embodiments, the thermal gradient maintained between the first lower temperature-regulating element and the second lower temperature-regulating element is in the range of 2° C. to about 10° C.; and wherein the thermal gradient maintained between the first lower temperature-regulating element and the second lower temperature-regulating element is in the range of 2° C. to about 10° C.

[0121] In some embodiments, the lower plate is modular. In some embodiments, the modular lower plate is transportable to the upper plate. In some embodiments, the upper plate includes an upper engagement surface complementary to the lower engagement surface and a cavity recessed relative to the upper engagement surface. In some embodiments, the upper plate is coupled to a force generating member. In some embodiments, the force generating member is configured to retract if pressure within the chamber exceeds a predetermined threshold.

[0122] In some embodiments, at least one of the upper plate or the lower plate is coupled to at least one of a motor, a piston, a spring, a screw mechanism, a lever, or a cam mechanism.

[0123] In some embodiments, the system further comprises at least one substrate transfer device. In some embodiments, the at least one substrate transfer device is selected from the group consisting of a gripper device, a forklift device, and a carrier transport device. In some embodiments, the system further comprises one or more substrate loading stations. BRIEF DESCRIPTION OF THE DRAWINGS

[0124] For a general understanding of the features of the present disclosure, reference is made to the drawings, in which like reference numerals are used throughout to identify like elements.

[0125] FIG. 1A illustrates a top view of a lower plate according to one embodiment of the present disclosure.

[0126] FIG. 1B illustrates a side view of a lower plate according to one embodiment of the present disclosure.

[0127] FIG. 1C illustrates a side view (longitudinal side) of a lower plate according to one embodiment of the present disclosure.

[0128] 1D shows a top view of a lower plate including vacuum ports, according to one embodiment of the present disclosure.

[0129] IE shows a top view of a lower plate including vacuum ports, according to one embodiment of the present disclosure.

[0130] 1F illustrates a perspective view of a lower plate including vacuum ports, according to one embodiment of the present disclosure.

[0131] 1G illustrates a top view of a lower plate including vacuum ports, according to one embodiment of the present disclosure.

[0132] 1H illustrates a top view of a lower plate including vacuum ports, according to one embodiment of the present disclosure.

[0133] FIG. 2A illustrates a top view of a lower plate according to one embodiment of the present disclosure.

[0134] 2B illustrates a side view of a first end of a lower plate according to one embodiment of the present disclosure.

[0135] 2C illustrates a side view of a second end of the lower plate according to one embodiment of the present disclosure.

[0136] FIG2D illustrates a side view (longitudinal side) of a lower plate according to one embodiment of the present disclosure.

[0137] FIG. 3A illustrates a top view of a lower plate according to one embodiment of the present disclosure.

[0138] FIG3B illustrates a side view (longitudinal side) of a lower plate according to one embodiment of the present disclosure.

[0139] FIG. 4A illustrates a top view of a lower plate according to one embodiment of the present disclosure.

[0140] 4B illustrates a side view of a first end of a lower plate according to one embodiment of the present disclosure.

[0141] 4C illustrates a side view of a second end of the lower plate according to one embodiment of the present disclosure.

[0142] FIG4D shows a side view (longitudinal side) of a lower plate according to one embodiment of the present disclosure.

[0143] 5A illustrates a sealing attachment removably coupled to a lower plate according to one embodiment of the present disclosure.

[0144] FIG. 5B illustrates a sealing attachment positioned above a lower plate, according to one embodiment of the present disclosure.

[0145] FIG. 6A illustrates a lower plate having a wedge-based shape according to one embodiment of the present disclosure.

[0146] 6B illustrates a lower plate having a wedge-based shape and an upper plate having a wedge-based shape, wherein the upper and lower plates are in contact with each other, according to one embodiment of the present disclosure.

[0147] 6C illustrates a side view of a first end of a lower plate having a wedge-based shape according to one embodiment of the present disclosure.

[0148] 6D illustrates a side view of a second end of a lower plate having a wedge-based shape according to one embodiment of the present disclosure.

[0149] 6E illustrates a side view of a lower plate having a wedge-based shape, according to one embodiment of the present disclosure.

[0150] 6F illustrates a side view of a lower plate having a wedge-based shape, according to one embodiment of the present disclosure.

[0151] FIG. 7A illustrates a lower plate coupled to a housing member according to one embodiment of the present disclosure.

[0152] 7B illustrates the lower plate coupled to the housing member according to one embodiment of the present disclosure.

[0153] FIG. 7C illustrates the lower plate coupled to the housing member, according to one embodiment of the present disclosure.

[0154] FIG. 7D illustrates the lower plate coupled to the housing member according to one embodiment of the present disclosure.

[0155] FIG. 8A illustrates a lower plate coupled to a heat sink according to one embodiment of the present disclosure.

[0156] FIG8B shows the lower plate coupled to a heat sink according to one embodiment of the present disclosure.

[0157] FIG. 9A illustrates a top view of an upper plate according to one embodiment of the present disclosure.

[0158] FIG. 9B illustrates a top view of an upper plate according to one embodiment of the present disclosure.

[0159] 9C illustrates a side view of a first end of an upper plate according to one embodiment of the present disclosure.

[0160] 9D illustrates a side view of a first end of an upper plate according to one embodiment of the present disclosure.

[0161] 9E illustrates a side view of a first end of an upper plate according to one embodiment of the present disclosure.

[0162] 9F illustrates a side view of a first end of an upper plate according to one embodiment of the present disclosure.

[0163] FIG. 9G illustrates an upper plate including a heating element according to one embodiment of the present disclosure.

[0164] FIG. 9H illustrates an upper plate including a heating element according to one embodiment of the present disclosure.

[0165] FIG. 10A shows a lower plate and an upper plate in contact with each other according to one embodiment of the present disclosure.

[0166] FIG. 10B shows a lower plate and an upper plate in contact with each other, according to one embodiment of the present disclosure.

[0167] FIG. 10C shows the lower plate and the upper plate in contact with each other, according to one embodiment of the present disclosure.

[0168] FIG. 10D shows the lower plate and the upper plate in contact with each other, according to one embodiment of the present disclosure.

[0169] FIG. 10E shows a lower plate and an upper plate in contact with each other, according to one embodiment of the present disclosure.

[0170] FIG. 10F shows the lower plate and the upper plate in contact with each other, according to one embodiment of the present disclosure.

[0171] FIG. 11A shows a lower plate with an integrated heat sink and an upper plate positioned above the lower plate, according to one embodiment of the present disclosure.

[0172] 11B shows a lower plate with an integrated heat sink and an upper plate in contact with the lower plate, according to one embodiment of the present disclosure.

[0173] 12A illustrates a top view of a lower plate movably coupled to a track according to one embodiment of the present disclosure.

[0174] 12B illustrates a side view of a lower plate movably coupled to a track, wherein the track is offset in a horizontal direction by a predetermined amount, according to one embodiment of the present disclosure.

[0175] FIG. 13A illustrates the relative positions to which the lower plate may move, according to one embodiment of the present disclosure.

[0176] FIG. 13B illustrates the relative positions to which the lower plate may move, according to one embodiment of the present disclosure.

[0177] 14A illustrates the progression of a lower plate from a first region to a second region and to a third region during processing, according to one embodiment of the present disclosure.

[0178] 14B illustrates a cam mechanism in communication with the upper plate and wherein the cam mechanism applies pressure to the upper plate (or structure disposed between the upper and lower plates) to apply or maintain sealing engagement therebetween, in accordance with an embodiment of the present disclosure.

[0179] 14C illustrates a cam mechanism in communication with the upper plate and wherein the cam mechanism is adapted to exert a force on the upper plate (or structure disposed therebetween) to impose or maintain sealing engagement between the upper and lower plates, in accordance with an embodiment of the present disclosure.

[0180] 14D illustrates a side view of an assembly including a force generating member, according to one embodiment of the present disclosure.

[0181] 14E illustrates a piston in communication with an upper plate and wherein the piston is adapted to exert pressure on the upper plate (or structure disposed between the upper and lower plates) to impose or maintain a sealing engagement between the upper and lower plates, in accordance with an embodiment of the present disclosure.

[0182] 14F illustrates a cam mechanism in communication with the upper plate and wherein the cam mechanism is adapted to exert a force on the upper plate (or structure disposed therebetween) to impose or maintain sealing engagement between the upper and lower plates, in accordance with an embodiment of the present disclosure.

[0183] 14G illustrates a piston in communication with an upper plate and wherein the piston is adapted to exert pressure on the upper plate (or structure disposed between the upper and lower plates) to impose or maintain a sealing engagement between the upper and lower plates, in accordance with an embodiment of the present disclosure.

[0184] 14H illustrates a cam mechanism in communication with the upper plate and wherein the cam mechanism is adapted to exert a force on the upper plate (or structure disposed therebetween) to impose or maintain sealing engagement between the upper and lower plates, in accordance with an embodiment of the present disclosure.

[0185] FIG. 141 illustrates multiple cam mechanisms coupled together by a single cam shaft, according to one embodiment of the present disclosure.

[0186] 14J illustrates a piston in communication with an upper plate and wherein the piston is adapted to exert pressure on the upper plate (or structure disposed between the upper and lower plates) to apply or maintain a sealing engagement therebetween, in accordance with an embodiment of the present disclosure.

[0187] 14K illustrates a cam mechanism in communication with the upper plate and wherein the cam mechanism is adapted to exert a force on the upper plate (or structure disposed therebetween) to impose or maintain sealing engagement between the upper and lower plates, in accordance with an embodiment of the present disclosure.

[0188] 14L illustrates a piston in communication with an upper plate and wherein the piston is adapted to exert pressure on the upper plate (or structure disposed between the upper and lower plates) to apply or maintain sealing engagement therebetween, in accordance with an embodiment of the present disclosure.

[0189] 14M illustrates a cam mechanism in communication with the upper plate and wherein the cam mechanism is adapted to exert a force on the upper plate (or structure disposed therebetween) to impose or maintain sealing engagement between the upper and lower plates, in accordance with an embodiment of the present disclosure.

[0190] 15A provides a top view of an assembly including a vessel in which a demasking operation may be performed, according to one embodiment.

[0191] 15B provides a side view of an assembly including a vessel and a hinged upper plate, according to one embodiment of the present disclosure.

[0192] 15C provides a perspective view of an assembly including a vessel in which a demasking operation may be performed, according to one embodiment.

[0193] 15D provides a perspective view of an assembly including a vessel in which a demasking operation may be performed, according to one embodiment.

[0194] 15E provides a perspective view of an assembly including a vessel in which a demasking operation may be performed, according to one embodiment.

[0195] 16A illustrates the progression of a lower plate from a first region to a second region and to a third region during processing, according to one embodiment of the present disclosure.

[0196] 16B illustrates the progression of a lower plate having a wedge-based shape from a first region to a second region and to a third region during processing, according to one embodiment of the present disclosure.

[0197] 16C illustrates the progression of a lower plate having a wedge-based shape from a first region to a second region and to a third region during processing, according to one embodiment of the present disclosure.

[0198] 16D illustrates a lower plate and an upper plate having a wedge-based shape and wherein the upper and lower plates can independently move toward each other, according to one embodiment of the present disclosure.

[0199] FIG17A illustrates a system with various interoperable modules according to one embodiment of the present disclosure.

[0200] FIG. 17B illustrates a system having four discrete components, according to one embodiment of the present disclosure.

[0201] FIG. 17C illustrates a system having multiple discrete components according to one embodiment of the present disclosure.

[0202] FIG. 18A illustrates components according to one embodiment of the present disclosure.

[0203] FIG. 18B illustrates components according to one embodiment of the present disclosure.

[0204] FIG. 18C illustrates an assembly according to one embodiment of the present disclosure.

[0205] 19A illustrates a top view of an assembly including a disc carousel, one or more substrate loaders, and a lower plate according to one embodiment of the present disclosure.

[0206] 19B illustrates a side view of the assembly of FIG. 19A including a carousel, one or more substrate loaders, and a lower plate, according to one embodiment of the present disclosure.

[0207] 19C illustrates a perspective view of the assembly of FIG. 19A including a carousel, one or more substrate loaders, and a lower plate, according to one embodiment of the present disclosure.

[0208] 20A shows a perspective view of a system including a plurality of carousels, substrate holders, a substrate loader, a lower plate, and an upper plate according to one embodiment of the present disclosure.

[0209] 20B illustrates a top view of a system including a plurality of carousels, substrate holders, a substrate loader, a lower plate, and an upper plate according to one embodiment of the present disclosure.

[0210] 21A illustrates the arrangement of heating and / or cooling elements relative to upper and lower plates according to one embodiment of the present disclosure.

[0211] 21B illustrates the placement of heating and / or cooling elements relative to upper and lower plates according to one embodiment of the present disclosure.

[0212] 21C illustrates the placement of heating and / or cooling elements relative to upper and lower plates according to one embodiment of the present disclosure.

[0213] 21D illustrates the arrangement of heating and / or cooling elements relative to upper and lower plates according to one embodiment of the present disclosure.

[0214] FIG22A shows a lower plate configured with two substrate tables according to one embodiment of the present disclosure, wherein the lower plate is complementary to the upper plate of FIG22B .

[0215] FIG. 22B illustrates an upper plate configured with two cavities, according to one embodiment of the present disclosure, wherein the upper plate is complementary to the lower plate of FIG. 22A .

[0216] 23A shows an upper plate and a lower plate positioned adjacent to each other, wherein the upper plate includes a deformable sealing element and the lower plate includes a tab, according to one embodiment of the present disclosure.

[0217] 23B illustrates an upper plate and a lower plate engaged with each other, wherein the upper plate includes a deformable sealing element and the lower plate includes a tab, according to one embodiment of the present disclosure.

[0218] 24A illustrates a side cross-sectional view of the body of a lower plate including one or more heating and / or cooling elements according to one embodiment of the present disclosure.

[0219] 24B illustrates a side cross-sectional view of the body of a lower plate including one or more heating and / or cooling elements, according to one embodiment of the present disclosure.

[0220] 24C illustrates a side cross-sectional view of the body (longitudinal side of the body) of a lower plate including one or more heating and / or cooling elements, according to one embodiment of the present disclosure.

[0221] 24D illustrates a side cross-sectional view of the body of a lower plate including one or more heating and / or cooling elements, according to one embodiment of the present disclosure.

[0222] 25A illustrates a side cross-sectional view of the body of a lower plate including one or more heating and / or cooling elements (eg, bores into which one or more heating modules (eg, heating cartridges) may be inserted) according to one embodiment of the present disclosure.

[0223] Figure 25B shows a side cross-sectional view of the body of a lower plate according to one embodiment of the present disclosure, which includes one or more heating and / or cooling elements (e.g., one or more fluid channels serving as conduits for a fluid, which can be heated and / or cooled, e.g., to a predetermined temperature, or heated and / or cooled on demand according to commands from a control unit).

[0224] 25C illustrates a side cross-sectional view of the body of the lower plate including one or more heating and / or cooling elements (eg, bores for inserting one or more heating modules; fluid channels), according to one embodiment of the present disclosure.

[0225] 25D illustrates a side cross-sectional view of the body of a lower plate including one or more heating and / or cooling elements (eg, multiple fluid channels), according to one embodiment of the present disclosure.

[0226] 25E illustrates a side cross-sectional view of the body of a lower plate including one or more heating and / or cooling elements (eg, thermoelectric modules), according to one embodiment of the present disclosure.

[0227] 25F illustrates a top cross-sectional view of the body of the lower plate including one or more heating and / or cooling elements (eg, bores for inserting one or more heating modules; fluid channels), according to one embodiment of the present disclosure.

[0228] 25G illustrates a top cross-sectional view of the body of a lower plate including one or more heating and / or cooling elements (eg, a network of fluid channels), according to one embodiment of the present disclosure.

[0229] 25H illustrates a top cross-sectional view of the body of a lower plate including one or more heating and / or cooling elements (eg, two separate fluid channel networks) according to one embodiment of the present disclosure.

[0230] 25I and 25J illustrate fluid flow through fluid channels in the lower plate according to one embodiment of the present disclosure.

[0231] 26A illustrates a side cross-sectional view of a body in communication with a thermal management module including one or more heating and / or cooling elements, according to one embodiment of the present disclosure.

[0232] 26B illustrates a side cross-sectional view of a main body in communication with a thermal management module, wherein both the main body and the thermal management module include one or more heating and / or cooling elements, according to one embodiment of the present disclosure.

[0233] 26C illustrates a side cross-sectional view of a body in communication with a thermal management module including one or more heating and / or cooling elements, according to one embodiment of the present disclosure.

[0234] 26D illustrates a side cross-sectional view of a main body in communication with a thermal management module, wherein both the main body and the thermal management module include one or more heating and / or cooling elements, according to one embodiment of the present disclosure.

[0235] 26E illustrates a side cross-sectional view of a main body in communication with a thermal management module, wherein both the main body and the thermal management module include one or more heating and / or cooling elements, according to one embodiment of the present disclosure.

[0236] 26F illustrates a sample processing device including a substrate supported by a body in thermal communication with a thermal management module, according to one embodiment of the present disclosure.

[0237] 26G illustrates a sample processing device including a substrate supported by a body in thermal communication with a thermal management module, according to one embodiment of the present disclosure.

[0238] FIG. 27A illustrates a body disposed within a carrier according to one embodiment of the present disclosure.

[0239] 27B illustrates a perspective view of a body inserted or embedded within a carrier according to one embodiment of the present disclosure.

[0240] FIG. 27C illustrates a perspective view of a body inserted or embedded within a carrier, according to one embodiment of the present disclosure.

[0241] Figure 27D shows a top view of a body inserted or embedded within a carrier according to one embodiment of the present disclosure.

[0242] 27E illustrates a side cross-sectional view of a body inserted or embedded within a carrier according to one embodiment of the present disclosure.

[0243] 27F illustrates a perspective view of a body inserted or embedded within a carrier in communication with a pickup member, according to one embodiment of the present disclosure.

[0244] 27G illustrates a perspective view of a body inserted or embedded within a carrier, wherein the carrier is in communication with a pickup member, and the body and carrier are in thermal communication with a thermal management module, according to one embodiment of the present disclosure.

[0245] 27H illustrates a perspective view of a body inserted or embedded within a carrier, wherein the carrier is in communication with a pickup member, and wherein the body and carrier are positioned over a thermal management module, according to one embodiment of the present disclosure.

[0246] 27I illustrates a forklift apparatus moving a carrier including a main body to a thermal management module according to one embodiment of the present disclosure.

[0247] 28A illustrates a top view of the body of a lower plate according to one embodiment of the present disclosure.

[0248] 28B illustrates a top view of the body of the lower plate according to one embodiment of the present disclosure.

[0249] 28C illustrates a perspective view of the body of the lower plate including a vacuum port and / or one or more indentations, according to one embodiment of the present disclosure.

[0250] Figure 28D shows the body of the lower plate including one or more indentations according to one embodiment of the present disclosure.This figure also depicts a gripper arrangement positioned adjacent the substrate.

[0251] Figure 28E shows the body of the lower plate including one or more indentations according to one embodiment of the present disclosure.The figure also depicts a gripper arrangement positioned adjacent the substrate.

[0252] Figure 28F shows the body of the lower plate including one or more indentations according to one embodiment of the present disclosure.This figure also depicts a gripper arrangement positioned adjacent the substrate.

[0253] 29A illustrates a side cross-sectional view of the body of an upper plate including one or more heating and / or cooling elements according to one embodiment of the present disclosure.

[0254] 29B illustrates a side cross-sectional view of the body of an upper plate including one or more heating and / or cooling elements, according to one embodiment of the present disclosure.

[0255] 29C illustrates a side cross-sectional view of the body of an upper plate including one or more heating and / or cooling elements (eg, fluid channels), according to one embodiment of the present disclosure.

[0256] 29D illustrates a side cross-sectional view of the body of an upper plate including one or more heating and / or cooling elements (eg, bores for inserting one or more heating modules; fluid channels), according to one embodiment of the present disclosure.

[0257] 29E illustrates a side cross-sectional view of the body of an upper plate including one or more heating and / or cooling elements (eg, bores for inserting one or more heating modules), according to one embodiment of the present disclosure.

[0258] 29F illustrates a top cross-sectional view of the body of an upper plate including one or more heating and / or cooling elements (eg, bores for inserting one or more heating modules; fluid channels), according to one embodiment of the present disclosure.

[0259] 29G illustrates a top cross-sectional view of the body of an upper plate including one or more heating and / or cooling elements (eg, a network of fluid channels), according to one embodiment of the present disclosure.

[0260] 30A shows a side cross-sectional view of a lower plate and an upper plate in communication with each other and with a chamber formed therebetween. This figure also shows that the upper plate and / or the lower plate include one or more heating and / or cooling elements according to one embodiment of the present disclosure.

[0261] FIG30B shows a side cross-sectional view of a lower plate and an upper plate in communication with each other and with a chamber formed therebetween. This figure also illustrates that, according to one embodiment of the present disclosure, the upper and / or lower plates include one or more heating and / or cooling elements (fluid channels; bores for inserting one or more heating modules).

[0262] 30C shows a side cross-sectional view of the lower plate and upper plate in communication with each other with a chamber formed therebetween. This figure also shows that the upper plate and / or the lower plate include one or more heating and / or cooling elements (fluid channels) according to one embodiment of the present disclosure.

[0263] Figure 30D shows a side cross-sectional view of the lower plate and upper plate in communication with each other, with a chamber formed therebetween. This figure also shows that the upper plate and / or the lower plate include one or more heating and / or cooling elements (fluid channels, thermoelectric modules) according to one embodiment of the present disclosure.

[0264] 30E and 30F each illustrate a fluid flow path between an upper plate and a lower plate, each of which includes one or more fluid channels.

[0265] 31A illustrates a sample processing assembly comprising a lower plate movably coupled to a subassembly, wherein the lower plate comprises a lower engagement surface and one or more substrate tables; an upper plate having an upper engagement surface complementary to the lower engagement surface; and a motorized vise, according to one embodiment of the present disclosure.

[0266] 31B shows a lower plate including a lower engagement surface and one or more substrate tables; a subassembly for transporting the lower plate; an upper plate having an upper engagement surface complementary to the lower engagement surface; and a jack-screw mechanism according to one embodiment of the present disclosure.

[0267] 31C shows a lower plate including a lower engagement surface and one or more substrate tables; a subassembly for transporting the lower plate; an upper plate having an upper engagement surface complementary to the lower engagement surface; and a scissor jack mechanism according to one embodiment of the present disclosure.

[0268] 31D shows a lower plate including a lower engagement surface and one or more substrate tables; a subassembly for transporting the lower plate; an upper plate having an upper engagement surface complementary to the lower engagement surface; and a scissor jack mechanism according to one embodiment of the present disclosure.

[0269] 31E shows a lower plate including a lower engagement surface and one or more substrate tables; a subassembly for transporting the lower plate; an upper plate having an upper engagement surface complementary to the lower engagement surface; and a scissor jack mechanism according to one embodiment of the present disclosure.

[0270] FIG. 32A illustrates a body with one or more attachment members according to one embodiment of the present disclosure.

[0271] FIG. 32B illustrates a body having a roughened or patterned surface according to one embodiment of the present disclosure.

[0272] FIG. 32C illustrates a body having a coating disposed thereon, according to one embodiment of the present disclosure.

[0273] FIG. 33A illustrates a gripper arm of a gripper device according to one embodiment of the present disclosure.

[0274] FIG33B illustrates a gripper arm of a gripper device according to one embodiment of the present disclosure.

[0275] FIG. 33C illustrates a gripper arm of a gripper device according to one embodiment of the present disclosure.

[0276] FIG33D illustrates a gripper arm of a gripper device according to one embodiment of the present disclosure.

[0277] FIG. 33E illustrates a gripper device including a suction cup according to one embodiment of the present disclosure.

[0278] 34A illustrates the movement of a substrate with a gripper device toward a sample processing assembly and the subsequent position of the substrate on the body of the lower plate of the sample processing assembly, according to one embodiment of the present disclosure.

[0279] 34B illustrates placing a substrate onto the body of a lower plate using a gripper device and gripper arms, according to one embodiment of the present disclosure.

[0280] FIG. 35A illustrates a forklift assembly according to one embodiment of the present disclosure.

[0281] FIG. 35B illustrates a forklift assembly according to one embodiment of the present disclosure.

[0282] FIG. 35C illustrates a forklift assembly according to one embodiment of the present disclosure.

[0283] FIG. 35D illustrates a forklift assembly according to one embodiment of the present disclosure.

[0284] 36A illustrates a forklift apparatus including cylinders acting on an upper surface of a base, according to one embodiment of the present disclosure.

[0285] 36B illustrates an alternative view of a forklift apparatus including cylinders acting on an upper surface of a base, according to one embodiment of the present disclosure.

[0286] 36C shows an alternative view of a forklift apparatus including cylinders acting on an upper surface of a base, according to one embodiment of the present disclosure.

[0287] 36D shows an alternative view of a forklift apparatus including cylinders acting on an upper surface of a base, according to one embodiment of the present disclosure.

[0288] 36E shows an alternative view of a forklift apparatus including cylinders acting on an upper surface of a base, according to one embodiment of the present disclosure.

[0289] FIG. 36F illustrates picking up a substrate using the forklift apparatus shown in FIG. 36A to FIG. 36E .

[0290] FIG. 37A shows a gripper arrangement for picking up and moving a carrier according to one embodiment of the present disclosure.

[0291] FIG. 37B shows a gripper arrangement for picking up and moving a carrier according to one embodiment of the present disclosure.

[0292] FIG. 37C shows a gripper arrangement for picking up and moving a carrier according to one embodiment of the present disclosure.

[0293] Figure 38A shows a substrate loading station with a platform and raised sides according to one embodiment of the present disclosure.

[0294] Figure 38B shows a substrate loading station with a platform and raised sides according to one embodiment of the present disclosure.

[0295] Figure 38C shows a substrate loading station having a platform and one or more spring mechanisms according to one embodiment of the present disclosure.

[0296] Figure 38D shows a substrate loading station having a platform and one or more spring mechanisms according to one embodiment of the present disclosure.

[0297] FIG38E illustrates a substrate loading station having a platform and one or more alignment grippers according to one embodiment of the present disclosure.

[0298] FIG38F illustrates a substrate loading station having a platform and one or more alignment grippers according to one embodiment of the present disclosure.

[0299] FIG38G illustrates a substrate loading station having a platform and one or more alignment grippers according to one embodiment of the present disclosure.

[0300] Figure 38H shows a substrate loading station having a platform and one or more rollers according to one embodiment of the present disclosure.

[0301] Figure 39A shows an upper plate and a lower plate connected to each other to form a chamber according to one embodiment of the present disclosure. The figure also shows four different thermal zones that can be independently adjusted so that at least a portion of the substrate or substrate table remains relatively the coolest component within the formed chamber.

[0302] Figure 39B shows an upper plate and a lower plate in communication with each other to form a chamber according to one embodiment of the present disclosure. The figure further shows five different thermal zones that can be independently adjusted so that at least a portion of the substrate or substrate table remains relatively cool within the formed chamber.

[0303] Figure 39C shows an upper plate and a lower plate connected to each other to form a chamber according to one embodiment of the present disclosure. The figure also shows six different thermal zones that can be independently adjusted so that at least a portion of the substrate or substrate table remains relatively the coolest component within the formed chamber.

[0304] 40A shows a top view of the lower plate showing different thermal zones A, B, B', C, and D. In some embodiments, at least a portion of thermal zone A is in communication with the substrate, and wherein at least a portion of the substrate is maintained at a lower temperature than thermal zones B, B', C, and / or D.

[0305] FIG40B illustrates a side cross-sectional view of an upper plate in communication with a lower plate, thereby forming a chamber, according to one embodiment of the present disclosure. The figure also illustrates that thermal zones A, B, C, D, E, and F can be independently adjusted to maintain at least a portion of a substrate disposed within the formed chamber at a temperature lower than that of at least thermal zones C, D, E, and F.

[0306] 40C shows a top view of the upper plate showing different thermal zones E and F. In some embodiments, at least a portion of thermal zone E is in communication with a substrate (e.g., a substrate disposed within a chamber formed by the upper and lower plates), and wherein at least a portion of the substrate is maintained at a lower temperature than thermal zones E and F.

[0307] 41A, 41B, and 41C each illustrate thermal analysis of an upper plate and / or lower plate in communication with one or more independently controllable heating and / or cooling elements.

[0308] 42A and 42B illustrate fluid channels in communication with the lower plate and other system components, which enables the fluid within the fluid channels to be cooled to a predetermined temperature.

[0309] Figures 43A-43H provide flow charts illustrating methods of performing demasking operations using any of the sample processing components and / or systems of the present disclosure.

[0310] FIG. 44 shows an example of times and temperatures used in the temperature ramp-up phase, the temperature hold phase, and the temperature ramp-down phase.

[0311] FIG. 45 provides an example of temperatures generated using a sample processing assembly according to one embodiment of the present disclosure.

[0312] FIG46 provides a schematic diagram illustrating one or more control systems communicatively coupled to one or more heating elements, one or more cooling elements, and one or more sensors. DETAILED DESCRIPTION

[0313] It should also be understood that, unless indicated to the contrary, in any method claimed herein that includes more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are presented.

[0314] definition

[0315] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "including" is defined as inclusive, e.g., "including A or B" means including A, B, or A and B.

[0316] As used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as being inclusive, e.g., including several elements or at least one element from a list of elements, but also including more than one element, and optionally including additional unlisted items. Only terms indicating the contrary, such as "only one" or "exactly one", or "consisting of..." as used in the claims, will refer to the inclusion of several elements or exactly one element from a list of elements. In general, the term "or" as used herein should be interpreted as indicating exclusive alternatives (e.g., "one or the other, but not both") only when preceded by an exclusive term such as "or", "one of", "only one", or "exactly one". "Consisting essentially of..." as used in the claims should have its ordinary meaning as used in the art of patent law.

[0317] The terms "including," "comprising," and "having" are used interchangeably and have the same meaning. Similarly, "s," "including," "having," etc. are used interchangeably and have the same meaning. Specifically, the definition of each term is consistent with the definition of "including" under ordinary U.S. patent law, and thus each term is to be understood as an open term meaning "at least the following" and is also to be interpreted as not excluding additional features, limitations, aspects, etc. Thus, for example, "a device having components a, b, and c" means that the device includes at least components a, b, and c. Similarly, the phrase: "a method involving steps a, b, and c" means that the method includes at least steps a, b, and c. Furthermore, although steps and processes may be outlined herein in a particular order, those skilled in the art will recognize that the order steps and processes may vary.

[0318] As used herein in the specification and claims, with respect to a list of one or more elements, the phrase "at least one" should be understood to mean at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of every element specifically listed in the list of elements, nor does it exclude any combination of elements in the list of elements. In addition to the elements specifically identified in the list of elements to which the phrase "at least one" refers, this definition also allows for the optional presence of other elements, whether or not related to the specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B," or equivalently, "at least one of A and / or B") can, in one embodiment, mean at least one optionally including one or more A but no B (and optionally including elements other than B); in another embodiment, mean at least one optionally including one or more B but no A (and optionally including elements other than A); in yet another embodiment, mean at least one optionally including one or more A and at least one optionally including one or more B (and optionally including other elements), and so on.

[0319] As used herein, the term "antigen" refers to a substance bound by an antibody, antibody analog (e.g., aptamer), or antibody fragment. Antigens can be endogenous, where they are produced intracellularly as a result of normal or abnormal cellular metabolism, or due to viral or intracellular bacterial infection. Endogenous antigens include xenogeneic (heterologous), autologous, and idiotypic or allogeneic (homologous) antigens. Antigens can also be tumor-specific antigens or presented by tumor cells. In this case, they are referred to as tumor-specific antigens (TSAs) and are typically produced by tumor-specific mutations. Antigens can also be tumor-associated antigens (TAAs), which are presented by tumor cells and normal cells. Antigens further include CD4+ antigens, which refer to any of a variety of cell surface markers expressed by leukocytes and can be used to distinguish cell lineages or developmental stages. Such markers can be recognized by specific monoclonal antibodies and numbered by their differentiation clusters.

[0320] As used herein, the term "fluid" refers to any liquid or liquid composition used in sample processing operations, including water, solvents, solutions (e.g., polar solvents, non-polar solvents), mixtures, colloids, suspensions, and the like. Such sample processing operations include adding a liquid or a composition containing a liquid to a substrate, a sample disposed on a substrate, and the like. Fluids can be aqueous or non-aqueous. Non-limiting examples of fluids include solvents and / or solutions used to dewax paraffin-embedded biological samples, aqueous detergent solutions, wash solutions, rinse solutions, acidic solutions, alkaline solutions, transfer solutions, and hydrocarbons (e.g., alkanes, isoalkanes, and aromatic compounds such as xylene). Further examples of fluids include solvents (and mixtures thereof) used to dehydrate or rehydrate biological samples. In some embodiments, wash solutions contain a surfactant to facilitate diffusion of the wash solution onto the sample-carrying surface of the slide. In some embodiments, acid solutions include deionized water, an acid (e.g., acetic acid), and a solvent. In some embodiments, alkaline solutions include deionized water, a base, and a solvent. In some embodiments, the transfer solution includes one or more glycol ethers, such as one or more propenyl glycol ethers (e.g., propylene glycol ether, dipropylene glycol ether, and tripropylene glycol ether), ethylene glycol-based glycol ethers (e.g., ethylene glycol ether, diethylene glycol ether, and triethylene glycol ether), and functional analogs thereof. Additional wash solutions, transfer solutions, acid solutions, and base solutions, as well as methods for applying them and apparatus for applying them, are described in U.S. Patent Application Publication No. 2016 / 0282374, the disclosure of which is incorporated herein by reference in its entirety.

[0321] As used herein, "horizontal" generally refers to an angle within about + / - 2 degrees of horizontal, for example, within about + / - 1 degree of horizontal, such as within about + / - 0.8 degrees of horizontal. Horizontal also refers to a small range of angles relative to horizontal, for example, an angle between about 0.1 degrees and 1.8 degrees relative to horizontal, such as an angle between about 0.2 degrees and about 1.2 degrees, for example, an angle between about 0.3 degrees and about 0.8 degrees. For example, a horizontally positioned or held substrate will have an orientation such that the substrate's major surface faces generally upward and downward and is substantially parallel to the ground. In certain embodiments, a horizontally held rectangular substrate, such as a microscope slide, will have an angle along its minor axis relative to horizontal of between about 0.0 degrees and about 2.0 degrees, and along its major axis relative to horizontal of between about 0.0 degrees and 2.0 degrees, with the substrate's major surface also facing generally upward and downward. Likewise, a horizontally arranged or positioned subassembly (eg, a track) is a subassembly that is substantially parallel to the ground or angled within approximately + / - 2 degrees from horizontal.

[0322] As used herein, the term "plurality" refers to two or more, eg, 3 or more, 4 or more, 5 or more, etc.

[0323] As used herein, the term "reagent" or "rare reagent" refers to a solution or suspension comprising a specific binding entity, an antibody (primary, secondary, or antibody conjugate), a nucleic acid probe (isolated nucleic acid or isolated synthetic oligonucleotide attached to a detectable label or reporter molecule), a demasking agent (defined herein), a detection probe, and a solution or suspension of a dye or staining molecule (e.g., H&E staining solution, Papanicolaou staining solution, etc.). The term "detection probe" refers to a nucleic acid probe or antibody that binds to a specific target (e.g., a nucleic acid sequence, a protein, etc.). The detection probe may include a label for detection, such as a radioisotope, an enzyme substrate, a cofactor, a ligand, a chemiluminescent or fluorescent agent, a hapten (including but not limited to DNP), and an enzyme. Alternatively, the detection probe may not contain a label or tag and may be detected indirectly (e.g., using a secondary antibody specific for the detection probe). The term "antibody conjugate" refers to an antibody conjugated (directly or indirectly) to one or more labels, wherein the antibody conjugate is specific for a specific target and wherein the label is detectable (directly or indirectly). For example, the antibody conjugate can be coupled to a hapten, such as through a polymer linker and / or a spacer, and the antibody conjugate can be detected indirectly by means of the hapten. As an alternative example, the antibody conjugate can be coupled to a fluorophore, such as through a polymer linker and / or a spacer, and the antibody conjugate can be detected directly. Examples of antibody conjugates are described in U.S. Publication No. 2014 / 0147906 and U.S. Patent Nos. 8,658,389; 8,686,122; 8,618,265; 8,846,320; and 8,445,191, the disclosures of which are incorporated herein by reference in their entirety.

[0324] As used herein, the term "slide" refers to any substrate of any suitable size on which a biological specimen may be placed for analysis (e.g., a substrate made in whole or in part of glass, quartz, plastic, silicon, etc.), and more particularly refers to a "microscope slide," such as a standard 3 x 1 inch microscope slide or a standard 75 mm x 25 mm microscope slide. Examples of biological specimens that may be placed on a slide include, but are not limited to, cytological smears, thin tissue sections (e.g., from a biopsy), and biological specimen arrays, such as tissue arrays, cell arrays, DNA arrays, RNA arrays, protein arrays, or any combination thereof. Thus, in one embodiment, a tissue section, DNA sample, RNA sample, and / or protein is placed on a specific location on a slide. In some embodiments, the term "slide" may refer to SELDI and MALDI chips, as well as silicon wafers.

[0325] As used herein, the term "specific binding entity" refers to a member of a specific binding pair. A specific binding pair is a pair of molecules that bind to each other to the substantial exclusion of binding to other molecules (e.g., the binding constant of the specific binding pair can be at least 10 greater than the binding constant of either member of the pair for other molecules in the biological sample). 3 M -1 , 10 4 M -1 or 10 5 M -1 Examples of specific binding moieties include specific binding proteins (e.g., antibodies, lectins, streptavidin, and avidin such as protein A). Specific binding moieties may also include molecules (or portions thereof) that are specifically bound by such specific binding proteins. Specific binding entities include the primary antibodies or nucleic acid probes described above.

[0326] As used herein, the terms "sample," "tissue sample," "specimen," or similar terms refer to any sample comprising biomolecules (e.g., proteins, peptides, nucleic acids, lipids, carbohydrates, or combinations thereof) obtained from any organism, including viruses. Examples of other organisms include mammals (e.g., humans; veterinary animals such as cats, dogs, horses, cows, and pigs; and laboratory animals such as mice, rats, and primates), insects, annelids, arachnids, marsupials, reptiles, amphibians, bacteria, and fungi. Biological samples include tissue samples (e.g., tissue sections and biopsies of tissue), cell samples (e.g., cytological smears, such as cervical smears or blood smears, or obtained by microdissection), or cell fractions, fragments, or organelles (e.g., obtained by lysing cells and separating their components by centrifugation or other means). Other examples of biological samples include blood, serum, urine, semen, feces, cerebrospinal fluid, interstitial fluid, mucus, tears, sweat, pus, biopsy tissue (e.g., obtained by surgical biopsy or needle biopsy), nipple aspirate, cerumen, breast milk, vaginal secretions, saliva, swabs (e.g., buccal swabs), or any material containing biomolecules and derived from a first biological sample. In certain embodiments, the term "biological sample" as used herein refers to a sample prepared from a tumor or a portion thereof obtained from a subject (e.g., a homogenized or liquefied sample).

[0327] As used herein, the terms "stain," "staining," or similar terms generally refer to any treatment of a biological specimen that detects and / or distinguishes the presence, location, and / or amount (e.g., concentration) of a specific molecule (e.g., lipid, protein, or nucleic acid) or a specific structure (e.g., normal or malignant cells, cytoplasm, nucleus, Golgi apparatus, or cytoskeleton) in the specimen. For example, staining can be used to compare a specific molecule or a specific cellular structure in a biological specimen with the surrounding tissue, and the intensity of the staining can be used to determine the amount of the specific molecule in the specimen. Stains can be used not only with brightfield microscopy but also with other observation tools such as phase contrast microscopy, electron microscopy, and fluorescence microscopy to aid in the visualization of molecules, cellular structures, and organisms. Some stains performed by the system allow for clear visualization of cellular outlines. Other stains performed by the system may rely on staining specific cellular components (e.g., molecules or structures) while leaving other cellular components unstained or staining them relatively minimally. Examples of various types of staining methods performed by the system include, but are not limited to, histochemistry, immunohistochemistry, and other methods based on intermolecular reactions (including non-covalent binding interactions) such as hybridization reactions between nucleic acid molecules. Staining methods include, but are not limited to, primary staining methods (such as H&E staining, cervical staining, etc.), enzyme-linked immunohistochemistry methods, and in situ RNA and DNA hybridization methods, such as fluorescence in situ hybridization (FISH).

[0328] As used herein, the term "substantially" refers to the qualitative condition of exhibiting the full or nearly full extent or degree of a characteristic or property of interest. In some embodiments, "substantially" means within about 20%. In some embodiments, "substantially" means within about 15%. In some embodiments, "substantially" means within about 10%. In some embodiments, "substantially" means within about 5%.

[0329] As used herein, the term "target" refers to any molecule whose presence, location, and / or concentration is or can be determined. Examples of target molecules include proteins, epitopes, nucleic acid sequences, and haptens, such as haptens covalently bound to proteins. Typically, a target molecule is detected using a conjugate of one or more specific binding molecules and a detectable label.

[0330] As used herein, the terms "unmask" or "unmasking" refer to the restoration of an antigen or target in fixed tissue and / or the improved detection of an antigen, amino acid, peptide, protein, nucleic acid, and / or other target. For example, it is believed that antigenic sites that might otherwise remain undetected may be revealed, for example, by disrupting some of the protein crosslinks surrounding the antigen during unmasking. In some embodiments, the antigen and / or other target is unmasked by applying one or more unmasking agents (defined below), heat, and / or pressure. In some embodiments, only one or more unmasking agents are applied to the sample to achieve unmasking (e.g., without the need for heat or pressure). In other embodiments, only heat is applied to achieve unmasking. In some embodiments, unmasking can occur solely in the presence of water and heat. Unmasking is further described in U.S. Patent Application Publication Nos. 2009 / 0170152 and 2009 / 0104654, the disclosures of which are incorporated herein by reference in their entirety. In some embodiments, demasking can begin once the demasking agent is applied to the sample, regardless of when and where the demasking operation occurs. For example, demasking can begin once the demasking agent is applied to the sample, but before the demasking operation begins.

[0331] As used herein, the term "demasking operation" refers to the step or stage of demasking performed within a sealed chamber. For example, the demasking operation may include the step of heating and / or pressurizing a sample disposed on a substrate in the presence of one or more demasking agents (defined below) for a predetermined amount of time within the sealed chamber. Other steps of the demasking operation may include cooling the chamber or any portion thereof or depressurizing the sealed chamber. Still other steps of the demasking operation include quenching, rapid boiling, and / or the step of dispensing additional fluid and / or reagents to the sample while in the sealed chamber. In some embodiments, the demasking operation may include a ramp-up phase, a temperature hold phase, a temperature ramp-down phase, a pressure ramp-up phase, a pressure hold phase, and / or a pressure ramp-down phase occurring after chamber formation.

[0332] As used herein, the term "demasking agent" refers to any liquid, including solutions and mixtures, that is dispensed into a sample to aid in demasking. In some embodiments, the demasking agent comprises a variety of components, such as those listed below. In some embodiments, the demasking agent is a buffered solution. In some embodiments, the buffered solution has a pH in the range of about 5 to about 10. In other embodiments, the buffered solution has a pH in the range of about 7 to about 9. In other embodiments, the buffered solution has a pH in the range of about 7.5 to about 11. Non-limiting examples of buffers include citric acid, potassium dihydrogen phosphate, boric acid, diethylbarbituric acid, piperazine-N,N'-bis(2-ethanesulfonic acid), dimethylarsinic acid, 2-(N-morpholino)ethanesulfonic acid, tris(hydroxymethyl)methylamine (TRIS), 2-(N-morpholino)ethanesulfonic acid (TAPS), N,N-bis(2-hydroxyethyl)glycine (Bicine), N-tris(hydroxymethyl)methylglycine (Tricine), 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES), 2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES), and combinations thereof. In some embodiments, the demasking agent is water. In other embodiments, the buffer solution can be composed of tris(hydroxymethyl)methylamine (TRIS), 2-(N-morpholino)ethanesulfonic acid (TAPS), N,N-bis(2-hydroxyethyl)glycine (Bicine), N-tris(hydroxymethyl)methylglycine (Tricine), 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES), 2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES), or a combination thereof.

[0333] In some embodiments, the demasking agent comprises a TRIS-based buffer having an alkaline pH. In some embodiments, the TRIS-based buffer has a pH of approximately 10 (e.g., at elevated temperature). In yet other embodiments, the buffer solution can be a sodium citrate phosphate buffer solution having a pH of approximately 6.0 at elevated temperature. In other embodiments, the demasking agent comprises approximately 0.05% citraconic anhydride. In other embodiments, the demasking agent comprises approximately 100 mM TRIS and has a pH between approximately 8 and approximately 10. In other embodiments, the demasking agent comprises approximately 10 mM citrate, approximately 2 mM EDTA, and approximately 0.05% Tween 20 and has a pH of approximately 6.2. In other embodiments, the demasking agent comprises approximately 0.01 M citrate buffer and has a pH of approximately 6.0.

[0334] In some embodiments, the demasking agent comprises a component that reacts with any released fixative to prevent it from reacting again with the sample. Examples of such demasking agents include purpald or dimedone. Alternatively or additionally, the demasking agent may comprise a component that reacts reversibly with the free amino groups of the protein and thereby protects them from reacting with any available formaldehyde, such as citraconic anhydride (CCA).

[0335] In other embodiments, the demasking agent comprises a chelating agent. Examples of chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), EGTA / AM (EGTA, tetra(acetoxymethyl ester)), (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) (BAPTA), BAPTA / AM (EGTA tetra(acetoxymethyl ester)), 5,5'-dimethyl-BAPTA-AM; 1,2-bis(2-amino-5-methylphenoxy)ethane (MAPTAM), N,N,N',N'-tetra(2-pyridylmethyl)ethane-1,2-diamine (TPEN), citrate, or ionophores such as ionomycin or calcimycin, or any combination or mixture thereof.

[0336] In other embodiments, the unmasking agent comprises an enzyme. Non-limiting examples of enzymes include proteases (such as trypsin, chymotrypsin, pepsin, or proteinase K), nucleases, glycanases, and hyaluronidase.

[0337] In other embodiments, the unmasking agent comprises a chaotropic agent. Non-limiting examples of chaotropic agents include butanol, ethanol, guanidine salts (such as guanidine hydrochloride or guanidine thiocyanate), lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, thiourea, and urea.

[0338] In other embodiments, the demasking agent comprises a nucleophile, such as a chemical that donates an electron pair to an electrophile. Non-limiting examples of nucleophiles include ammonia, primary amines, secondary amines, and tertiary amines. Other examples of nucleophiles include hydrazines, alcohols, and halides.

[0339] In other embodiments, the demasking agent comprises a Lewis acid. Non-limiting examples of Lewis acids include metal ions such as iron (III) ions, aluminum ions, magnesium ions, or other electron-deficient compounds such as toluenesulfonic acid, boric acid, boron trifluoride, and tartaric acid.

[0340] In other embodiments, the demasking agent may include a surfactant. As used herein, "surfactants" are classified as anionic, cationic, or nonionic, depending on their chemical mode of action. Typically, surfactants reduce the interfacial tension between two liquids. An example of a surfactant is sodium lauryl sulfate. Examples of suitable nonionic surfactants include polyethylene glycol monocetyl ether, cetearyl alcohol, cetyl alcohol, cocamide diethanolamine, cocamide monoethanolamine, decyl glucoside, octylphenoxypolyethoxyethanol, polyethylene glycol monoisocetyl ether, lauryl glucoside, nonylphenoxypolyethoxyethanol, 4-nonylphenyl-polyethylene glycol, 1-(4-nonylphenyl)-1,4,7,10,13,16,19,22,25-nonaoxaheptacosan-27-ol, nonoxynol ether, glyceryl laurate, octaethylene glycol monododecyl ether, oleyl alcohol, polyethylene glycol-polypropylene glycol, polyglyceryl polyricinoleate, polysorbate, and sorbitan monostearate. Sorbitan tristearate; stearyl alcohol; polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether; and polyoxyethylene (20) sorbitan monooleate. Octyl-, decyl-, dodecyl-glucopyranoside, -maltoside or deoxycholic acid. Exemplary surfactants are sold under the following names: Brij® 35, TWEEN®, Tergitol™, Triton™, Ecosurf™, Dowfax™, Polysorbate 80™, BigCHAP, deoxyBigCHAP, IGEPAL®, soap essence, Thesit®, Nonidet®, Pluronic F-68, digitonin, deoxycholate, etc. Specific disclosed working examples involve the use of a surfactant selected from Brij® 35, TWEEN®, Tergitol™, Triton™. Additional antigen unmasking agents are disclosed in US Patent No. 8,486,335, the disclosure of which is incorporated herein by reference in its entirety.

[0341] In some embodiments, the demasking agent comprises deionized water, TRIS, and a chelating agent. In some embodiments, the demasking agent comprises deionized water, TRIS, and a chelating agent and has a pH in the range of about 7 to about 9.5. In some embodiments, the demasking agent comprises deionized water, TRIS, a chelating agent, and a preservative. In some embodiments, the demasking agent is CCl, available from Entana Medical Systems, Inc., Tucson, AZ, USA. Other demasking agents are further described herein in U.S. Patent Application Publication No. 2009 / 0170152, the disclosure of which is incorporated herein by reference in its entirety.

[0342] Overview

[0343] As described herein, the present disclosure relates to a sample processing assembly comprising (a) a lower plate and (b) an upper plate that is complementary to the lower plate. The "complementary" upper and lower plates each include features or sets of features that complement each other. For example, the lower plate may have a first set of features (e.g., a substrate table and a lower engagement surface), and the upper plate may have a second set of features (e.g., a cavity and an upper engagement surface). In this example, the cavity of the upper plate may be complementary to the substrate table; and the upper engagement surface may be complementary to the lower engagement surface.

[0344] In some embodiments, the sample processing assembly is configured such that the lower plate and the upper plate can contact each other to form a chamber, such as a sealed chamber. In some embodiments, one or both of the lower plate and the upper plate are movable. In some embodiments, the lower plate and the upper plate are independently movable, such as independently movable in any one of the x-coordinate, y-coordinate, and z-coordinate directions. In other embodiments, one of the lower plate or the upper plate is movable, while the other is fixed. In some embodiments, the lower plate moves toward the fixed upper plate. In other embodiments, the upper plate moves toward the fixed lower plate.

[0345] In some embodiments, one or both of the lower plate and / or the upper plate include one or more independently operable heating and / or cooling elements. In some embodiments, any heating and / or cooling elements in the upper plate can be operated in conjunction with those heating and / or cooling elements in the lower plate. As further described herein, any independently operable heating and / or cooling elements in the upper plate and / or the lower plate can also be operated in conjunction with one or more thermal management modules. As a result, by independently controlling the various heating and / or cooling elements present in the upper and lower plates (or within one or more thermal management modules), the temperature of the sample (such as a sample disposed on a substrate), the substrate, and / or the chamber can be controlled.

[0346] For example, various heating and / or cooling elements present in the upper and lower plates (or those in the thermal management module) can be independently operated so that at least a portion of the substrate or a sample disposed on the substrate is maintained as the coolest structure within the chamber. For example, the substrate or a sample disposed on the substrate can be maintained at a lower temperature than the upper plate, the lower plate, ports, valves, chamber walls, and / or any other structure within the chamber formed by the upper and lower plates.

[0347] In some embodiments, one or more independently operable heating and / or cooling elements in either the lower and / or upper plates may be arranged such that, during operation, their placement and / or positioning allow one or more temperature gradients to be established within the formed chamber (or along any component of the formed chamber, including the lower and upper plates). In some embodiments, the one or more heating and / or cooling elements are arranged such that, during operation, a temperature gradient is established between the substrate table and those portions of the chamber surrounding the substrate table. In this regard, the temperature gradient enables the substrate table (or at least a portion of a substrate disposed thereon) to be maintained at a relatively lower temperature than other adjacent areas of the chamber. In some embodiments, the temperature gradient is established and maintained during all stages of the demasking operation (e.g., heating, pressurization, cooling, decompression, quenching, etc.). In some embodiments, the temperature gradient is established and maintained such that a portion of the sample or substrate is the "coldest" component within the formed chamber. For example, a portion of the sample and / or substrate may be maintained at a temperature lower than any other portion of the upper and / or lower plates, chamber walls, nozzles, ports, temperature probes, pressure sensors, etc.

[0348] In some embodiments, the upper plate is coupled to one or more force-generating components. In some embodiments, the one or more force-generating components are selected from motors, screws, levers, pistons, cam mechanisms, and the like. In some embodiments, the one or more force-generating components apply a predetermined amount of force to at least the upper plate or any structure interposed therebetween. In some embodiments, the predetermined amount of force applied is less than the force generated by a predetermined threshold pressure within the chamber formed by the complementary upper and lower plates. In this regard, the predetermined force applied by the force-generating component is less than the force pushing the upper and lower plates apart, and thus, the force-generating component can retract or slide, allowing pressure to be released from the formed chamber. Alternatively, the predetermined amount of force applied does not exceed the force generated by the predetermined threshold pressure within the chamber. This is believed to serve as a safety mechanism, mitigating pressure buildup within the chamber that exceeds the predetermined threshold pressure. In some embodiments, the lower and / or upper plates may further include one or more ports and / or valves to allow pressure exceeding the predetermined threshold pressure to be released from the chamber's internal environment. For example, a spring with a specific spring constant may be used as at least part of the force-generating component, such that when the internal pressure of the chamber exceeds the predetermined pressure, the spring further collapses, releasing pressure from the chamber.

[0349] The present disclosure also relates to a sample processing assembly comprising a chamber, such as a chamber formed by a lower plate and an upper plate complementary to the lower plate. In some embodiments, demasking (e.g., antigen repair, target repair) can be performed within the chamber. For example, gas and / or steam can be introduced into the chamber to pressurize the chamber. For another example, one or more heating elements can be activated to heat one or more fluids and / or reagents present in the chamber. In some embodiments, one or more cooling elements are activated simultaneously with the one or more heating elements to enable uniform heating of the substrate, a sample disposed on the substrate, or the chamber itself. In some embodiments, the chamber can have any size and / or shape. In some embodiments, the chamber is sized to accommodate at least a portion of the substrate and any sample, fluid, and / or reagent disposed thereon.

[0350] The present disclosure also relates to systems comprising one or more sample processing components. In some embodiments, the system comprises one or more sample processing components and at least one dispensing device. In some embodiments, the system further comprises one or more liquid removal modules, a mixing module, a pressurization module, an imaging module, a coverslip module, and the like. In some embodiments, the system comprises a control system comprising one or more processors and one or more memories.

[0351] Components of the sample processing assembly

[0352] As described above, the sample processing assembly can include a lower plate and an upper plate complementary to the lower plate. In some embodiments, the sample processing assembly further includes at least one additional component, such as a subassembly, a support member, a substrate loader, a substrate holder, a force generating member, etc. Each of these components will be further described herein.

[0353] Lower plate

[0354] The sample processing assembly of the present disclosure includes at least one lower plate, such as those shown in Figures 1-4 or a modular lower plate, such as those shown in Figures 26A-26E.

[0355] 1A-1C , in some embodiments, the lower plate 10 includes a body 16 having a lower engagement surface 11 and one or more substrate stages 12. In some embodiments, the one or more substrate stages 12 each include an upper surface 12C (e.g., an upper planar surface) adapted to support a substrate 15 (e.g., a microscope slide).

[0356] In some embodiments, the one or more substrate tables 12 are elevated relative to the lower engaging surface 11. In other embodiments, the one or more substrate tables 12 are recessed relative to the lower engaging surface 11. In other embodiments, the one or more substrate tables 12 include a first portion that is elevated or flush relative to the lower engaging surface 11 and a second portion that is recessed or flush relative to the lower engaging surface 11.

[0357] In some embodiments, the substrate stage 12 has a substantially rectangular shape (see, e.g., FIG. 1A or FIG. 1D ). In other embodiments, the substrate stage 12 has rounded edges. In still other embodiments, the substrate stage 12 has a beveled edge 172 (see, e.g., FIG. 1H ). In some embodiments, the substrate stage 12 is approximately the size of the substrate. In other embodiments, the substrate stage 12 is larger than the substrate. In still other embodiments, the substrate stage 12 is smaller than the substrate. For example, the substrate stage 12 may be smaller than a standard microscope slide in at least one dimension. In some embodiments, a substrate stage sized smaller than the substrate allows any condensate formed within the chamber formed by the lower plate and the complementary upper plate to form or migrate beneath the slide. In some embodiments, at least a portion of one edge of the substrate overhangs a side of the substrate stage.

[0358] In some embodiments and as described herein, the lower plate 10 is configured to complement the upper plate 30 (see, e.g., FIG. 6B ). In some embodiments, the lower plate 10 is adapted to maintain a level position of the substrate 15 supported by the substrate stage 12 as the lower plate 10 moves. In this manner, any fluids and / or reagents disposed thereon remain on the substrate; for example, they do not migrate to the edges of the substrate or flow off the substrate. In some embodiments, the body 16 of the lower plate 10 is unitary. In other embodiments, the body 16 comprises two or more coupled components. These and other features of the lower plate 10 are described herein.

[0359] In some embodiments, the body 16 of the lower plate 10 can have any size or shape, such as a polygonal shape or a wedge-based shape. Similarly, the lower engagement surface 11 can have any size or shape, as long as it is complementary to the upper engagement surface 31 of the upper plate 30 described herein (see, for example, FIG. 9A ). In some embodiments, the lower engagement surface 11 of the lower plate 10 can have the same general shape as the lower plate. Although not depicted, in some embodiments, the lower plate 10 can have a lower engagement surface that is not a straight line but rather a curved line, again assuming that any upper engagement surface of the upper plate is complementary to the curved lower engagement surface.

[0360] In some embodiments, and as shown in any of Figures 1, 2, 3, and 4, the body 16 of the lower plate 10 can have a generally rectangular shape. In other embodiments, and referring to Figure 1A, the body 16 of the lower plate 10 comprises a length 2 and a width 3, the overall proportions of which conform to those of a microscope slide. In embodiments where the body 16 has a rectangular shape, the plane formed by the upper planar surface 12C of the substrate stage 12 is parallel to the plane formed by the lower engagement surface 11.

[0361] In other embodiments, the body 16 of the lower plate 10 has a wedge-based shape. For example and at least as Figure 6A As shown, one side of the body 16 can have a wedge-like shape that tapers from approximately a first height 4 to approximately a second height 5. FIG6C and FIG6D illustrate front views of the ends of the wedge-like body 16, respectively. In some embodiments, the body 16 having a wedge-like shape can further include a lower engagement surface 11 and a substrate stage 12 ( FIG6A ). In some embodiments and as shown in FIG6A , the substrate stage 12 is configured such that a plane formed by the upper planar surface 12C of the substrate stage 12 (and any substrate 15 supported by the substrate stage 12) intersects a plane formed by the lower engagement surface 11. In some embodiments, the upper surface 12C of the substrate stage 12 is configured such that a substrate disposed thereon is held horizontally. For example, a substrate disposed on the upper surface 12C will remain parallel to the ground as a lower plate, coupled to the subassembly as described herein, traverses the subassembly.

[0362] Referring to Figures 6A and 6B , in some embodiments, the first end 12A of the substrate table can be elevated relative to the first portion 11A of the lower engagement surface, while the second end 12B of the substrate table can be flush with or recessed relative to the second portion 11B of the lower engagement surface. In some embodiments and referring to Figure 6A , the upper surface 12C of the substrate table is disposed at a height 6 that is the same as, higher than, or lower than the height 4. Figure 6E provides an alternative wedge-shaped body 16, in which the first end 12A of the substrate table is elevated relative to the first portion 11A of the lower engagement surface, while the second end 12B of the substrate table is recessed relative to the second portion 11B of the lower engagement surface. Figure 6F provides yet another alternative lower plate 10 having a wedge-shaped body 16.

[0363] Although not shown in FIG6A , the substrate stage 12 can include one or more alignment members, such as those described further herein. In some embodiments, the height of the one or more alignment members is less than the height of a substrate disposed on the substrate stage. In other embodiments, the height of the one or more alignment members is approximately half the height of the substrate, or less than half the height of the substrate. Furthermore, the body 16 of the wedge-shaped lower plate 10 can include a groove, such as a groove surrounding the periphery of the substrate stage 12. In some embodiments, the groove is adapted to at least partially engage a sealing body, such as those described further herein.

[0364] In some embodiments, the substrate stage 12 may include a vacuum port 170. In some embodiments, the substrate stage 12 includes a vacuum port and one or more vacuum sealing members 171, such as shown in Figures 1D to 1H. In some embodiments, the vacuum port 170 extends vertically through the body 16 and is in fluid communication with one or more vacuum lines and / or a vacuum source. In some embodiments, the one or more vacuum sealing members 171 have a non-circular shape. In some embodiments, the one or more vacuum sealing members 171 are made of a material such as rubber, silicone, or fluorocarbon rubber. In some embodiments, the one or more vacuum sealing members 171 include an O-ring. In some embodiments, the substrate stage includes one vacuum sealing member. In other embodiments, the substrate stage includes two or more vacuum sealing members. In other embodiments, the substrate stage includes three or more vacuum sealing members. In some embodiments, each of the one or more vacuum sealing members is adjacent to each other.

[0365] In some embodiments, the one or more vacuum sealing members 171 are compressible and / or temporarily deformable (e.g., compressible by approximately 5%, compressible by approximately 10%, compressible by approximately 15%, compressible by approximately 20%, compressible by approximately 25%, etc.). In some embodiments, the one or more vacuum sealing members 171 can be in an uncompressed state, wherein the one or more vacuum sealing members 171 can extend upwardly beyond the surface 12C of the substrate stage 12. In some embodiments, the one or more vacuum sealing members 171 can be in a compressed state, wherein the one or more vacuum sealing members 171 can be configured to maintain an airtight seal with the back side of a substrate (e.g., the back side of a microscope slide), such that the substrate is drawn against the surface 12C of the substrate stage 12 by a vacuum drawn through a vacuum port 170.

[0366] In some embodiments, a vacuum can be drawn between the substrate and the substrate table. For example, sufficient vacuum can be drawn to inhibit or limit movement of the substrate along the substrate table. The vacuum can be reduced or eliminated to remove the substrate from the substrate table. Any of the vacuum ports, sealing members, sealing surfaces, vacuum lines, and vacuum sources described in U.S. Patent No. 9,989,448 (the disclosure of which is incorporated herein by reference in its entirety) can be used within the present disclosure. Furthermore, any of the methods for operating the vacuum ports described in U.S. Patent No. 9,989,448 can be applied to the lower plate 10 of the present disclosure.

[0367] With reference to at least Figures 2A-2D, in some embodiments, the substrate stage 12 may further include one or more alignment members 14. In some embodiments, the one or more alignment members 14 are adapted to guide the substrate to an appropriate position on the upper surface 12C of the substrate stage 12, for example, to a position centered within the upper surface 12C of the substrate stage 12. In some embodiments, the substrate stage 12 may include between one and ten alignment members. In some embodiments, the alignment members 14 are protrusions that protrude perpendicularly from the surface of the substrate stage 12. In some embodiments, the protrusions can have any size and shape, such as cylindrical or polygonal. In some embodiments, the alignment members 14 are pins that protrude from the upper surface 12C of the substrate stage 12. In some embodiments, the one or more alignment members 14 are located near one or both of the ends 12A and 12B of the substrate stage. In some embodiments, the height of the one or more alignment members is less than the height of the substrate. In other embodiments, the height of the one or more alignment members is approximately half the height of the substrate, or less than half the height of the substrate.

[0368] In the case of a microscope slide supported by a substrate stage 12, one or more alignment members 14 can be positioned on the substrate stage 12 such that they are positioned at the distal ends of the microscope slide. For example, one, two, or three alignment members 14 can be positioned on the substrate stage 12 at the marked end 15A of the microscope slide 15; and another one, two, or three alignment members 14 can be positioned on the substrate stage 12 at the opposite distal end (e.g., the specimen-carrying end) of the slide 15. In some embodiments, one or more alignment members 14 can be positioned along the periphery of the longitudinal length of the substrate stage 12. For another example, and again in the case of a microscope slide, one or more alignment members 14 can be positioned on the substrate stage 12 along each longitudinal edge of the slide supported by the substrate stage 12. In some embodiments, the alignment members 14 can be spaced equidistantly from one another. In other embodiments, the alignment members 14 can be randomly spaced.

[0369] Referring at least to Figures 3A and 3B, in some embodiments, the lower plate 10 may further include a groove 13 that at least partially surrounds the main body 16 of the lower plate 10. As shown in Figures 3A and 4A, in some embodiments, the groove 13 may surround the substrate stage 12, such that the lower plate 10 includes a first lower engagement surface 11A and a second lower engagement surface 11B. In some embodiments, the groove 13 is configured such that a sealing body can removably engage a groove wall (not shown). In some embodiments, the sealing body inserted into the groove 13 may have a first portion that at least partially engages the groove wall; and a second portion that protrudes from the groove 13 and extends at least partially above the engagement surfaces 11A and / or 11B (such that it can be used to communicate with the upper engagement surface 31). In some embodiments, the sealing body is in the form of an O-ring seal. "O-ring seal" refers to an annular seal, regardless of its cross-sectional shape. In some embodiments, the substrate stage 12 may further include one or more alignment members, as shown in Figures 4A-4C.

[0370] In some embodiments, the sealing body can be made of a chemically and / or heat-resistant material. In some embodiments, the sealing body is made of metal. In some embodiments, the sealing body is compressible and / or temporarily deformable. In some embodiments, the sealing body is made of materials such as rubber, silicon or fluorocarbon rubber. Suitable examples of rubber include but are not limited to ethylene propylene diene monomer (EPDM), ethylene propylene rubber, chloroprene rubber (CR), butyl rubber (IIR) and silicone rubber. Suitable examples of fluorocarbon rubber include but are not limited to binary system vinylidene fluoride rubbers such as vinylidene fluoride / hexafluoropropylene copolymers, vinylidene fluoride / chlorotrifluoroethylene copolymers and vinylidene fluoride / pentafluoropropylene copolymers, ternary system vinylidene fluoride rubbers such as vinylidene fluoride / tetrafluoroethylene / hexafluoropropylene copolymers, vinylidene fluoride / tetrafluoroethylene / perfluoroalkyl vinyl ether copolymers and vinylidene fluoride / tetrafluoroethylene / propylene copolymers, tetrafluoroethylene / propylene copolymers, tetrafluoroethylene / perfluoroalkyl vinyl ether copolymers and thermoplastic fluorocarbon rubbers.

[0371] In other embodiments, the seal body may be constructed from a perfluoroelastomer, such as those described in U.S. Patent No. 7,834,096, the disclosure of which is incorporated herein by reference in its entirety. In other embodiments, the seal body may be constructed from nitriles, such as acrylonitrile-butadiene (NBR), hydrogenated nitrile (HNBR), and carboxylated nitrile (XNBR); fluorocarbons (FKM); fluorosilicone (FVMQ); perfluoroelastomers (FFKM); tetrafluoroethylene propylene (FEPM); ethylene acrylate (AEM); polyacrylate; and thermoplastic polyurethane. In other embodiments, the seal may be constructed from a heat-resistant material, such as any of the materials described in U.S. Patent No. 7,919,554, the disclosure of which is incorporated herein by reference in its entirety.

[0372] In some embodiments, the sealing body 18 is integrated into the removable sealing attachment 17, such as shown in FIG5B. In some embodiments, the removable sealing attachment 17 is configured to engage a portion of the lower plate 10 (however, the removable sealing attachment 17 can also be configured to engage a portion of the upper plate 30). For example, the removable sealing attachment 17 may include one or more clips 24 that facilitate attachment to a portion of the lower plate 10 (see, e.g., FIG5B). Figure 5B ). In other embodiments, the removable sealing attachment 17 can frictionally engage the periphery of the lower plate 10. In still other embodiments, the removable sealing attachment 17 can be screwed into the lower plate 10. In some embodiments, the sealing body 18 of the removable sealing attachment 17 fits into the groove 13 of the lower body and at least partially engages the walls of the groove 13. In some embodiments, at least a portion of the sealing body 18 of the removable sealing attachment 17 extends above the upper surface of the removable sealing attachment 17. In some embodiments, the sealing body is positioned so that it can contact a portion of the upper engaging surface 31. In some embodiments, the removable sealing attachment 17 includes an upper surface 25 that serves as an engaging surface, such as a lower engaging surface for contacting and / or facilitating formation of a seal with the upper engaging surface 31 of the upper plate 30. In some embodiments, the removable sealing attachment 17 helps maintain the seal formed between the upper and lower plates, particularly when the chamber formed by the upper and lower plates is pressurized. In some embodiments, the removable sealing attachment 17 includes a cavity 26 so that when the removable sealing attachment 17 is coupled to the lower plate 10 , the substrate stage 12 (and any substrate 15 supported by the substrate stage 12 ) can be unmasked for further processing.

[0373] In some embodiments, the lower plate 10 includes one or more indentations within the body 16 adjacent to the substrate table 12. For example, Figures 28A, 28B, and 28C each illustrate a body 16 having two indentations 173A and 173B, where the one or more indentations are adjacent to two longitudinal sides of the substrate table 12. Indentations 173A and 173B can be of any size and / or shape. Furthermore, indentations 173A and 173B can have any depth within the body 16. In some embodiments, the two indentations 173A and 173B are aligned with each other. In other embodiments, the two indentations 173A and 173B are offset from each other. In some embodiments, the indentations are also provided within the substrate table 12 itself.

[0374] In some embodiments, the one or more indentations are sized to accommodate a gripper device, such as an arm sized to accommodate a gripper device suitable for picking up and moving a substrate. Suitable gripper devices for picking up slides supported by a substrate table are described herein. Figures 28D and 28E illustrate the body 16 of the lower plate 10 including one or more indentations 173A and 173B, and further illustrate a gripper device 460 having notched ends 462A and 462B within gripper arms 461A and 461B, respectively. These figures illustrate the notched ends 462A and 462B inserted into the indentations 173A and 173B within the body 16. In the configuration shown in FIG28E , the gripper arms 461A and 461B are in an extended configuration, but the gripper arms 461A and 461B can be retracted, allowing the gripper arms 461A and 461B to move in the directions X and X' shown. As the gripper arms 461A and 461B retract, the notched ends 462A and 462B of the gripper 460 move toward the substrate 15 and at least partially around the edge of the substrate 15, allowing it to be picked up and moved by the gripper device 460, such as in the Z direction shown. In some embodiments, the indentations 173A and 173B in the body 16 are sized to accommodate at least the notched ends 462A and 462B in both the extended and retracted positions of the gripper arms 461A and 461B.

[0375] The lower plate may include one or more heating and / or cooling elements 21. In some embodiments, the one or more heating and / or cooling elements 21 are embedded within the body 16 of the lower plate 10. For example, Figures 24A and 24C each illustrate a lower plate 10 having a body 16 with one or more heating and / or cooling elements 21 embedded within the body 16 of the lower plate 10. Any number of heating and / or cooling elements may be embedded within the body 16 of the lower plate, for example, one or more heating and / or cooling elements, two or more heating and / or cooling elements, three or more heating and / or cooling elements, four or more heating and / or cooling elements, five or more heating and / or cooling elements, six or more heating and / or cooling elements, etc. Similarly, the body 16 of the lower plate 10 can include any combination of heating and / or cooling elements, such as 1 heating and / or cooling element; 1 heating element and 1 cooling element; 2 heating elements; 2 cooling elements; 2 heating elements and 1 cooling element; 2 cooling elements and 1 heating element; 2 cooling elements and 2 heating elements; 3 heating elements; 3 cooling elements; 3 heating elements and 1 or 2 cooling elements; 3 cooling elements and 1 or 2 heating elements; 3 cooling elements and 3 heating elements; etc.

[0376] In some embodiments, the one or more heating and / or cooling elements 21 can have any arrangement within the body 16 of the lower plate. For illustrative purposes only, FIG. 24D illustrates one or more heating elements 21A arranged in parallel with one or more cooling elements 21B. However, skilled artisans will appreciate that the one or more heating and / or cooling elements can be arranged side by side (e.g., heating elements 401A and 401B in FIG. 25A ), in a staggered arrangement (e.g., compare heating elements 401A and 401B with heating elements 411A and 411B), evenly spaced, randomly spaced, etc. In some embodiments, the one or more heating and / or cooling elements can be positioned below the substrate table. In other embodiments, the one or more heating and / or cooling elements can be positioned below the substrate table, with additional heating and / or cooling elements positioned adjacent to the one or more heating and / or cooling elements positioned below the substrate table, with the additional heating and / or cooling elements flanking the one or more heating and / or cooling elements positioned below the substrate table (see FIG. 25C , FIG. 25D , and FIG. 25F ).

[0377] In some embodiments, the one or more heating and / or cooling elements can be arranged such that during their operation (e.g., during any heating and / or cooling steps, temperature ramp-up or ramp-down steps), their arrangement and / or cooling positioning relative to the main body of the lower plate 10 enables the establishment of a temperature (or thermal) gradient between different portions of the main body 16 of the lower plate 10. In some embodiments, the one or more heating and / or cooling elements are arranged such that, during operation, a temperature gradient is established between the substrate table and those portions of the main body surrounding the substrate table. In this regard, the temperature gradient enables the substrate table (or at least a portion of a substrate disposed thereon) to be maintained at a relatively lower temperature than other adjacent regions of the main body 16 of the lower plate 10. For example, and referring to FIG. 40A , the surface 12C of the substrate table 12 can be maintained at temperature A, while those portions of the main body 16 of the lower plate 10 surrounding or adjacent to the substrate table 12 can be maintained at temperatures B, B', C, and D. Following this particular example, temperature gradients may be established between A and B, between A and B', between A and C, and between A and D. In some embodiments, when temperature A is maintained at a temperature lower than any of temperatures B, B', C, and D, the surface 12C of the substrate table 12 will be the relatively "coldest" portion of the lower plate 10.

[0378] In some embodiments, a temperature gradient is established and maintained during a temperature ramp-up phase (e.g., heating) and during a temperature ramp-down phase (e.g., cooling or reducing the amount of heat supplied). In some embodiments, even before the temperature ramp-up phase begins, the temperature of the substrate table is maintained relatively cooler than surrounding and / or adjacent portions of the body 16. It is believed that establishing and maintaining the temperature gradient even during the temperature ramp-up and ramp-down phases can keep at least a portion of the substrate relatively cooler than other portions of the body of the lower plate. In this regard, when the lower and upper plates (described herein) are in communication with each other so as to form a chamber therebetween (e.g., forming a chamber enclosing a substrate and / or substrate table), at least a portion of the substrate and / or substrate table is maintained as the "coldest" component within the formed chamber, e.g., cooler than any other portion of the upper and / or lower plates, including any nozzles, ports, temperature probes, pressure sensors, etc.

[0379] For example, the one or more heating and / or cooling elements can be independently operated such that during any temperature ramp phase, the surface 12C of the substrate table 12 is consistently maintained at a temperature lower than the temperature of surrounding and / or adjacent regions of the body 16. That is, at any particular time during the temperature ramp phase, in some embodiments, the substrate table 12 is maintained at a temperature lower than the temperature of surrounding and / or adjacent portions of the body 16. For example, and referring to FIG40A , assuming the temperature ramp occurs over a 10-minute period, if a temperature measurement were taken at time 3 minutes, temperature A would be relatively lower than temperatures B, B', C, and / or D. Similarly, if a second temperature measurement were taken at time 7 minutes, temperature A would be relatively lower than temperatures B, B', C, and / or D. Once any of portions A, B, B', C, and / or D reaches a predetermined temperature (e.g., at time 10 minutes), the temperature measurement for A would again be relatively lower than temperatures B, B', C, and / or D.

[0380] As described in further detail herein, this is believed to enable any substrate supported by the substrate table (or any portion of the substrate) to have a relatively lower temperature than adjacent portions of the main body of the lower plate. In some embodiments, the temperature gradient between the substrate table and the portions of the main body 16 located on either side of the substrate table is in a range between 5°C and 50°C. In other embodiments, the temperature gradient between the substrate table and the portions of the main body 16 located on either side of the substrate table is in a range between 5°C and 40°C. In still other embodiments, the temperature gradient between the substrate table and the portions of the main body 16 located on either side of the substrate table is in a range between 5°C and 30°C. In further embodiments, the temperature gradient between the substrate table and the portions of the main body 16 located on either side of the substrate table is in a range between 5°C and 25°C. In yet further embodiments, the temperature gradient between the substrate table and the portions of the main body 16 located on either side of the substrate table is in a range between 5°C and 20°C. In other embodiments, the temperature gradient between the substrate table and the portions of the main body 16 located on either side of the substrate table is in a range between 5°C and 15°C. In other embodiments, the temperature gradient between the substrate table and the portions of the body 16 on either side of the substrate table is in a range between 5°C and 10°C.

[0381] The one or more heating and / or cooling elements embedded within the body 16 of the lower plate 10 can be of any type known to those skilled in the art, including any of those described herein. Those skilled in the art will appreciate that, in those lower plate embodiments that include more than one heating and / or cooling element, the same or different types of heating and / or cooling elements can be used. By way of example only, the body 16 of the lower plate 10 can include one or more fluid channels for circulating a fluid to achieve heating and / or cooling of the body, and the body 16 can also include one or more bores into which one or more heating modules (e.g., heating cartridges) can be inserted. As another example, the body 16 of the lower plate 10 can include a thermoelectric cooling module to enable cooling to temperatures below room temperature, as well as one or more fluid tubes or channels for circulating a fluid having a temperature above room temperature.

[0382] In some embodiments, the one or more heating elements 21 comprise a heating cartridge. Suitable heating cartridges include those described in U.S. Patent Nos. 3,927,301, 4,617,455, 1,882,365, and 1,433,691. In some embodiments, the heating cartridge comprises a heating foil curved around a cylindrical core, such as a ceramic core, which is completely covered by a metal cylinder.

[0383] The body 16 of the lower plate 10 may include one or more bores into which a heating module, such as a heating cartridge, may be inserted. In some embodiments, any number of bores may be provided within the body 16 of the lower plate 10, such as 1, 2, 3, 4, 6, 8, 12, etc. The bores may be arranged in any manner within the body 16 of the lower plate. Thus, the body 16 may accommodate 1, 2, 3, 4, 6, 8, 12, etc. heating cartridges. For example, FIG. 25A and FIG. Figure 25C Each shows a body 16 having two bores 401A and 401B into which the heating cartridges can be inserted.

[0384] In some embodiments, the one or more bores may be evenly spaced from one another. In other embodiments, the one or more holes may be randomly spaced. In some embodiments, the bores open onto the longitudinal sides of the lower plate 10 (see, e.g., FIG. Figure 1D , which shows a longitudinal side of the lower plate 10). Alternatively, in other embodiments, the bore may be open on the "short side" of the lower plate, as shown in FIG. 25A . As shown in FIG. 25F , the bore may extend across the entire length of the body 16 of the lower plate (see bore 401B) or may extend only partially through the body 16 of the lower plate 10 (see bores 401A and 401C). The bores themselves may independently have any size and / or diameter.

[0385] In some embodiments, each of the one or more heating cartridges inserted into the body 16 can be independently operated. For example, assuming that the body 16 of the lower plate 10 includes three heating cartridges inserted into each of three different bores, each of the three heating cartridges can be independently operated, such as heating cartridges 1 and 3 can be operated at 80% of their rated power, while cartridge 2 can be operated at 40% of its rated power.

[0386] In other embodiments, the one or more heating elements 21 embedded in the body 16 comprise ceramic elements having an electrically conductive ceramic material sintered into a shell of insulating ceramic material. In other embodiments, the one or more heating elements 21 embedded in the body 16 comprise resistive electric heaters. In still other embodiments, the one or more heating elements 21 embedded in the body 16 comprise induction coils. In other embodiments, the one or more heating elements embedded in the body 16 utilize one of infrared radiation or microwave radiation to heat the substrate or a sample disposed on the substrate. In some embodiments, the infrared radiation or microwave radiation is used to heat a fluid, such as a fluid present in a reservoir or on a substrate. In these embodiments, the fluid is heated with the infrared radiation or microwave radiation, causing steam to be generated. In some embodiments, the generated steam is used to heat the sample and / or pressurize the chamber formed by the lower plate and the complementary upper plate, as further described herein.

[0387] In other embodiments, the one or more heating elements embedded within the body 16 are selected from one or more of a heating foil, a heating wire, or a heating tape. Further embodiments illustrating the use of heating and / or cooling elements within the upper and lower plates are shown in FIG. 21A - FIG. 21D . In some embodiments, any heating and / or cooling elements within the lower plate 10 can operate in conjunction with those within the upper plate 30 or any thermal management module. Consequently, those skilled in the art will appreciate that by controlling the various heating and / or cooling elements present in the upper and lower plates and / or thermal management modules, the temperature of the substrate table, any substrate supported by the substrate table, and / or the chamber can be adjusted, for example, to maintain at least a portion of a sample or at least a portion of a substrate as the coolest component within any chamber formed by the upper and lower plates.

[0388] In some embodiments, the one or more heating or cooling elements 21 embedded within the body 16 comprise a Peltier device or a thermoelectric module 402. Suitable Peltier devices include those described in U.S. Patent Nos. 4,685,081, 5,028,988, 5,040,381, and 5,079,618, the disclosures of which are incorporated herein by reference in their entireties. For example, FIG. 25E illustrates a thermoelectric module embedded within the body 16 of the lower plate 10, wherein the "hot side" of the thermoelectric module is in thermal communication with the body 16 of the lower plate 10. Of course, the "cold side" of the thermoelectric module can similarly be in thermal communication with the body 16 of the lower plate 10.

[0389] In other embodiments, the one or more heating and / or cooling elements 21 embedded within the body 16 include one or more fluid channels for circulating a fluid within the body 16 of the lower plate 10. In some embodiments, the fluid is heating oil. In other embodiments, the fluid is a mixture, such as an aqueous mixture including a polyol (e.g., polyethylene glycol or polypropylene glycol) and / or an alcohol (e.g., ethanol or methanol).

[0390] The one or more fluid channels embedded within the body 16 of the lower plate can have any configuration and can operate independently. For example, and referring to Figures 25B and 25F, the one or more fluid channels 411A and 411B can be arranged as two separate parallel conduits that independently allow a suitable fluid to flow through the body 16, for example, through first openings (414A and 414B) on one side of the body 16, through fluid channels 411A and 411B, and out of second openings (413A and 413B) on the opposite side of the body 16. In this particular example, fluid channels 411A and 411B can each be configured for cooling, each for heating, or one for heating while the other for cooling. Similarly, fluid channels 411A and 411B, while both configured for heating, can be configured for heating at different temperatures.

[0391] FIG25D , which illustrates a cross-section of the body 16, shows another example of the body 16 of the lower plate 10 including a plurality of fluid channels 411A-411D. In this particular embodiment, the plurality of fluid channels 411A-411D can each be an independent fluid channel, all of which can be interconnected, or some (e.g., channels 411B and 411C) can be interconnected while others are not. In some embodiments, some of the fluid channels 411A-411D can be used to circulate fluid to heat the body 16, substrate stage 12, substrate 15, or any sample deposited on substrate 15, while other fluid channels 411A-411D can be used to circulate fluid to cool the body 16, substrate stage 12, substrate 15, or any sample deposited on substrate 15. In some embodiments, the one or more fluid channels are in communication with one or more fluid delivery lines, ports, fluid reservoirs, heating devices, cooling devices, heat exchanges, pumps, and / or valves, allowing fluid to be independently supplied to each fluid channel.

[0392] Alternatively, the one or more fluid channels may comprise a network of fluid channels 412, such as those shown in FIG. 25G . The network of fluid channels 412 may include one or more independently controllable inlets 414, outlets 413, or valves (not shown) to direct circulation along a specific fluid flow path or provide circulation only within a portion of the channel network. In some embodiments, the network of fluid channels may be in communication with a fluid reservoir (not shown), where the fluid reservoir may be heated or cooled as needed. In some embodiments, the lower plate includes more than one network of fluid channels. For example, FIG. 25H illustrates a lower plate 10 comprising a first network of fluid channels 412A and a second network of fluid channels 412B. Each of the networks of fluid channels 412A and 412B can operate independently, for example, each may contain fluids heated to different temperatures, each may have different fluid flow rates, etc. FIG. 25I and FIG. 25J both illustrate a lower plate 10 having at least one fluid channel 411 disposed therein, wherein the fluid channel is in communication with a recirculation pump 601 and a chiller 602. In some embodiments, the fluid channel 411 is further connected to a fluid reservoir.

[0393] In some embodiments, the substrate or a sample disposed on the substrate can be heated with steam, such as described herein. In these embodiments, heating and / or cooling elements within the lower plate can be used for thermal management in conjunction with introducing steam into the sample disposed on the substrate.

[0394] In some embodiments, the lower plate may include one or more cooling elements. In some embodiments, the one or more cooling elements are active cooling elements. For example, the active cooling element may include one or more fluid channels to facilitate the flow of a coolant and thereby provide active cooling of the sample, the upper plate, and / or the lower plate, as described above.

[0395] In other embodiments, the one or more cooling elements are passive cooling elements, such as heat sinks. In some embodiments, the lower plate 10 includes a heat sink. In some embodiments, the heat sink is integral with the lower plate 10. In some embodiments, the heat sink is formed within the body 16 of the lower plate. In other embodiments, the heat sink is formed within a housing member 20 that is engageable with the body 16, such as described further herein. For example, as shown in Figures 8A and 8B, the heat sink 29 can be integral with the housing member 20. In other embodiments, the heat sink can be fixedly secured to the bottom surface of the lower plate 10, for example, by being glued, screwed, or clamped to the body 16 or housing member 20. In some embodiments, the heat sink is a liquid-cooled heat sink, or the heat sink includes one or more fans, thermoelectric coolers, or any combination thereof.

[0396] As described above, in those embodiments where the body 16 of the lower plate 10 includes two or more heating and / or cooling elements 21, the two or more heating and / or cooling elements 21 may be the same or different. For example, FIG. 25C and FIG. Figure 25F Each shows the body 16 of the lower plate 10 including two different types of heating and / or cooling elements. In these specific examples, the body 16 includes two fluid channels 411A and 411B (which can be interconnected and / or independent of each other) and two bores 401A and 401B into which heating cartridges can be inserted. In this particular embodiment, the two fluid channels can be used independently to heat and / or cool the body 16, the substrate stage 12, the substrate 15, or any sample deposited on the substrate 15.

[0397] In some embodiments, the two independently operable fluid channels 411A and 411B can be used to establish a first temperature (e.g., 95°C), while the two independently operable heating cartridges within bores 401A and 401B can be used to establish a second temperature (e.g., 140°C). In this manner, a temperature gradient can be established between portions of the main body 16 of the lower plate 10. Further following the above example, due to the arrangement of the various heating and / or cooling elements and the temperatures achieved by each of the various heating and / or cooling elements, the portion of the main body 16 below the substrate stage 12 can have a lower temperature than the temperature established at the periphery of the main body 16. According to this example, also due to the established temperature gradient, the substrate 15 supported by the substrate stage 12 can have a relatively lower temperature than the periphery of the main body.

[0398] In some embodiments, the lower plate 10 may include a device for mixing fluids and / or reagents, disposed on the surface of the substrate 15. In some embodiments, the device for mixing fluids and / or reagents is a non-contact mixing device. In some embodiments, the device for non-contact mixing of fluids and / or reagents may be integrated into the body of the lower plate. In other embodiments, the device for non-contact mixing of fluids and / or reagents may be coupled to the body of the lower plate. In some embodiments, the lower plate 10 may include or communicate with an element capable of imparting vibrations to the substrate 15 supported by the substrate stage 12. In other embodiments, the lower plate 10 may include an acoustic wave generator, such as a transducer. In some embodiments, the transducer is a mechanical transducer. In other embodiments, the transducer is a piezoelectric transducer. In some embodiments, the transducer is comprised of a piezoelectric wafer that generates mechanical vibrations. In some embodiments, surface transducers are used to distribute or mix fluid volumes on a glass slide. Suitable apparatus and methods for contactless mixing are described in PCT Publication No. WO / 2018 / 215844, the disclosure of which is incorporated herein by reference in its entirety.

[0399] In some embodiments, the lower plate 10 (or the main body 16 , or the housing member 20 ) is formed by machining or milling a solid block of material. In other embodiments, the lower plate 10 (or the main body 16 , or the housing member 20 ) is formed by a 3D printing process. In other embodiments, the lower plate 10 or any portion thereof is produced from a mold. The lower plate 10 can be made of any material. For example, the lower plate 10 or any component thereof can be made of a metal or alloy; ceramic; glass; a glass-epoxy laminate; or a plastic (e.g., derived from a polymer, copolymer, or polymer or copolymer blend). Examples of suitable polymers include, but are not limited to, polyetheretherketone, polyimide, polyetherimide, polytetrafluoroethylene, polysulfone, polyvinylidene fluoride, and polyphenylene sulfide. Examples of suitable metallic materials include aluminum and steel. In some embodiments, if the lower plate is made of metal, the metal can be coated or uncoated (e.g., coated with a fluoropolymer).

[0400] In some embodiments, the lower plate has a total mass in the range of about 30 grams to 200 grams. In other embodiments, the lower plate has a total mass in the range of about 40 grams to 180 grams. In still other embodiments, the lower plate has a total mass in the range of about 40 grams to 160 grams. In still other embodiments, the lower plate has a total mass in the range of about 40 grams to 140 grams. In other embodiments, the lower plate has a total mass in the range of about 40 grams to 120 grams. In other embodiments, the lower plate has a total mass in the range of about 50 grams to 120 grams. In other embodiments, the lower plate has a total mass in the range of about 50 grams to 100 grams.

[0401] In some embodiments, the lower plate has a heat capacity ranging from about 40 J / C to about 110 J / C. In some embodiments, the lower plate has a heat capacity ranging from about 40 J / C to about 100 J / C. In some embodiments, the lower plate has a heat capacity ranging from about 40 J / C to about 90 J / C. In some embodiments, the lower plate has a heat capacity ranging from about 50 J / C to about 80 J / C. In some embodiments, the lower plate has a heat capacity ranging from about 50 J / C to about 70 J / C. In some embodiments, the lower plate has a heat capacity ranging from about 55 J / C to about 75 J / C.

[0402] With reference to at least Figures 7A-7D, 8A, and 8B, in some embodiments, the lower plate 10 includes a body 16 and a housing member 20. As described above, in some embodiments, the body 16 of the lower plate 10 and the housing member 20 are integral with one another and formed from a single, unitary piece, e.g., milled or machined from a single, unitary piece. In other embodiments, the body 16 of the lower plate 10 is adapted to fit (e.g., releasably fit) within the housing member 20 (see Figures 7A-7D and 8B). In alternative embodiments, the body 16 of the lower plate 10 is fixedly attached to the housing member 20, e.g., glued, screwed, or clamped thereto. In other embodiments, the body 16 of the lower plate 10 is removably engaged with the housing member 20.

[0403] For example, the body 16 of the lower plate 10 can be inserted into and removed from the housing member 20 as needed. Herein, a single type of lower plate 10 can be designed and used with various types of housing members 20, wherein the various housing members can have different shapes and / or designs (e.g., a housing member including an integral heat sink; a housing member including one or more heating and / or cooling elements without an integral heat sink). In some embodiments, the housing member 20 is configured to receive the body 16 such that the upper surface 12C of the substrate table 12 is recessed relative to the upper surface 20C of the housing member 20 ( FIGS. 7A and 8B ).

[0404] In other embodiments, the housing member 20 is configured to receive the body 16 such that the upper surface 12C of the substrate table 12 is flush with the upper surface 20C of the housing member 20 ( FIG. 7B ). In still other embodiments, the housing member 20 is configured to receive the body 16 such that the upper surface 12C of the substrate table 12 is raised relative to the upper surface 20C of the housing member 20 ( FIG. 7C ). In other embodiments, as shown in FIG. 7D , the housing member 20 includes one or more heating and / or cooling elements 21 in thermal communication with the body 16. These one or more heating and / or cooling elements 21 may include any combination of those described herein. Referring again to FIG. 7D , the one or more heating and / or cooling elements 21 may be sandwiched between the body 16 of the lower plate 10 and the housing member 20. Alternatively, the one or more heating and / or cooling elements may be separately provided in one or both of the body 16 of the lower plate 10 and / or the housing member 20.

[0405] In some embodiments, the lower plate 710 has a modular design (see, for example, FIG. 26A-26E ). It is believed that the modular design of the lower plate enables the lower plate (or any portion thereof) to be used with a variety of different sample processing assemblies 100 and for a variety of different purposes in a variety of different ways. For example, the modular lower plate 710 can be adapted to rest on, engage, or couple to other components of the sample processing assembly 100.

[0406] In some embodiments, the modular lower plate 710 includes a body 716 having a lower engagement surface 711. In some embodiments, the modular lower plate 710 includes a thermal management module (described herein). In some embodiments, the body 716 can rest on, engage, or couple to the thermal management module 440. FIG. 26 , for example FIG. 26A - Figure 26G Each shows the body 716 resting on the thermal management module 440 .

[0407] In some embodiments, the body 716 includes a substrate stage 712 that is integral with and part of the body 716. In some embodiments, the body 716 can be picked up, transported, and deposited onto the thermal management module 440, with the substrate supported by the substrate stage of the body 716 (which, together with the substrate stage 712 and the body 716 of the thermal management module 440, constitute the modular lower plate 710). In some embodiments, the body 716 can be transported between different thermal management modules 440 or between different components of the sample processing assembly 100 or system 200. In some embodiments, the body 716 itself can include any of the components described herein with respect to the body 16 of the lower plate 10, such as one or more heating and / or cooling elements, one or more alignment members, and the like. The body 716 can be constructed of any material, including any of those described herein with respect to the lower plate 10.

[0408] In other embodiments, the main body 716 includes a lower engagement surface but does not include an integral substrate stage. Instead, the modular lower plate 710 includes the main body 716 with a detachable substrate stage. In this manner, the detachable substrate stage can serve as a substrate carrier. For example, a substrate can be placed on the detachable substrate stage, and the substrate and detachable substrate stage pair can be picked up, transported, and deposited onto the main body 716 together. Further following this example, once a demasking operation is completed using the modular lower plate 710 (e.g., including a separate substrate stage, the main body 716, and a thermal management module), the detachable substrate stage (again serving as a substrate carrier) can be picked up and transported to the sample processing assembly 100 or other components of the system 200. For example, the detachable substrate and detachable substrate stage can be transported together to a staining apparatus or coverslipping apparatus. In some embodiments, the substrate and separate substrate stage pair remain together throughout all processing steps in the system 200.

[0409] In some embodiments, and referring to FIG26A , the modular lower plate 710 includes a body 716 having a substrate platform 712 and a lower engagement surface 711, wherein the body 716 is in thermal communication with the thermal management module 440. In some embodiments, the thermal management module 440 includes one or more heating and / or cooling elements 21, including any of those described herein. For example, the thermal management module 440 can include any number of heating and / or cooling elements, such as one heating element, one cooling element, one heating element and one cooling element, two heating elements and one cooling element, two heating elements, two cooling elements, two heating elements and two cooling elements, and so forth. In some embodiments, the thermal management module 440 can be used to maintain and establish any of the temperature gradients described herein. In some embodiments, both the body 716 and the one or more thermal management modules 440 include one or more heating and / or cooling elements 21 (see, for example, FIG26B ).

[0410] In some embodiments, and with reference at least to FIG. 26C and FIG. 26D , thermal management module 440 includes one or more electrical contacts and / or one or more ports, such as one or more electrical contacts for supplying power to another system component and / or one or more ports for supplying fluid to a fluid channel or for receiving fluid from a reservoir. Similarly, body 716 of modular lower plate 710 may include one or more complementary electrical contacts for receiving power from thermal management module 440 and / or one or more complementary ports for receiving fluid from thermal management module 440.

[0411] For example, and as shown in FIG26C , the body 716 of the modular lower plate 710 can include one or more bores 401A and 401B into which the heater cartridge is inserted. To maintain the ability of the body 716 to be transported between different processing areas and / or different sample processing assemblies 100 (or even other components of the system 200) without being hindered by electrical wiring connected to the body 716 or its subcomponents, the body 716 can include one or more electrical contacts 442A and 442B to enable power to be received from the thermal management module 440. Similarly, and as shown in FIG26C and FIG26D , the body 716 can include one or more fluid channels 411 , wherein fluid can be received into the fluid channels 411 via one or more ports 441A and 441B, which are complementary to the one or more ports on the thermal management module 440. In this way, the main body 716 of the modular lower plate 710 can be moved without being directly connected to any fluid delivery lines.

[0412] FIG26E illustrates another embodiment in which the body 716 is in thermal communication with the thermal management module 440. The body 716 of the modular lower plate 710 includes bores 401A and 401B into which heater cartridges can be inserted, and the body 716 of the modular lower plate 710 includes electrical contacts 442A and 442B for providing power to the heater cartridges inserted into the bores 401A and 401B. The embodiment shown in FIG26E further illustrates that the thermal management module 440 may include one or more independent fluid channels 411A and 411B. In this particular modular lower plate 710 configuration, power can be delivered from the thermal management module 440 via the electrical contacts 442A and 442B to the heater cartridges inserted into the bores 401A and 401B to achieve heating of the body 716 of the modular lower plate 710 and / or the substrate stage 712. At the same time, the thermal management module 440 can provide additional heating to the body 716 of the modular lower plate 710 and / or the substrate stage 712; or can provide cooling to the body 716 of the modular lower plate 710 and / or the substrate stage 712 based on the temperature of the fluid circulating in the fluid channels 411A and 411B.

[0413] FIG26F shows substrate 15 positioned on a substrate table (not shown) that is integral with a body 716. In this particular embodiment, body 716 is shown in thermal communication with a thermal management module 440, which includes one or more heating and / or cooling elements. FIG26F also shows body 716 in communication with a transport member 433, enabling transport member 433 to deliver body 716 to a position above thermal management module 440 but below upper plate 30. FIG26G shows substrate table 712 integral with body 716 of modular lower plate 710, wherein body 716 further includes a lower engagement surface 711, vacuum port 170, and vacuum sealing element 171. Body 716 of modular lower plate 710 is shown supported by and in thermal communication with thermal management module 440.

[0414] refer to Figure 32A , the body 716 may further include one or more attachment members 431A and 431B positioned on opposite ends (e.g., longitudinal ends) of the body 716. In some embodiments, the one or more attachment members 431A and 431B facilitate movement of the body 716 by one or more grippers or other devices, the grippers or other devices being configured to pick up and / or move the body 716, such as in any of the x-coordinate direction, the y-coordinate direction, and the z-coordinate direction. For example, a gripper or other picking device may be configured to pick up and move the body 716 of 710 by holding onto the first attachment member 431A and the second attachment member 431B. In still other embodiments, the body 716 may include one or more recesses 430A and 430B in the lower surface of the body 716 so that a gripper or picking device (further described herein) can gently lift the body 716 from below, thereby supporting it during any transport operation (see Figure 32A ).

[0415] In some embodiments, one or more sides of the body 716 have been roughened or patterned 160 (see Figure 32B ) to increase friction between the one or more sides of the body 716 and a gripper or other pickup device configured to grasp the body 716. In other embodiments, one or more sides of the body 716 have a surface that includes a coating 161 (see FIG. 32C ), such as a polymer coating, to increase friction between the one or more coated sides of the body 716 of 710 and a gripper or other pickup device configured to grasp the body 716.

[0416] As shown in Figures 27A-27E , a body 716 having a substrate table 712 and a lower engagement surface 711 can be incorporated into the carrier block 432. The carrier block 432 can have any size or shape. In some embodiments, the size and / or shape of the carrier block facilitates its pickup by the gripper assembly 460 or other pickup device. Figure 27D shows a bottom view of the body 716 incorporated into the carrier block 432. Figure 27E shows a side cross-sectional view of the body 716 incorporated into the carrier block 432.

[0417] In some embodiments, the body 716 can be fixed within the carrier block 432. In other embodiments, the body 716 can be removably coupled to the carrier block 432. In some embodiments, a single body 716 can be used with carrier blocks 432 of varying sizes and / or shapes. In some embodiments, the carrier block 432 is disposable, while the body 716 is reusable. In some embodiments, and referring to FIG. 27A , the body 716 further includes one or more attachment members 431A and 431B. In some embodiments, the one or more attachment members 431A and 431B are protrusions extending from the body 716 (e.g., from a longitudinal end of the body) and integral with the body 716. In some embodiments, the attachment members 431A and 431B on the body 716 can have any size and / or shape, as long as the carrier block 432 has a complementary shape to accommodate the attachment members 431A and 431B. In some embodiments, the one or more attachment members 431A and 431B facilitate alignment of the body 716 within the carrier block 432 and / or secure the body 716 within the carrier block 432 when the combined body 716 and carrier block 432 is moved, such as to the thermal management module 440 or other components of the sample processing assembly 100 or system 200.

[0418] In some embodiments, the carrier block 432 is composed of a material selected from polymers, copolymers, metals, and the like. In some embodiments, the carrier block 432 is composed of the same material as the body 716. In other embodiments, the body 716 is composed of a first material (e.g., metal) while the carrier block 432 is composed of a second material (e.g., a heat-resistant copolymer). In some embodiments, one or more sides of the carrier block 432 have a roughened or patterned surface to increase friction between the one or more sides of the carrier block 432 and a gripper or other pickup device. In other embodiments, one or more sides of the carrier block 432 have a surface comprising a coating, such as a polymer coating, to increase friction between the one or more coated sides of the carrier block 432 and a gripper or other pickup device.

[0419] The movement of the body 716 comprising the carrier block 432 is illustrated in Figures 27F-27H. Specifically, Figure 27F shows the body 716 comprising the carrier block 432 in communication with the carrier pickup member 450. In some embodiments, the carrier pickup member 450 comprises a carrier pickup body 452 and a pair of carrier pickup arms 451A and 451B. In some embodiments, the carrier pickup body 452 and the carrier pickup arms 451A and 451B are configured to support the edges of the carrier block 432. For example, the carrier block 432 can be sized to rest on a portion of the carrier pickup body 452 and on top of both the carrier pickup arms 451A and 451B. Figure 27G shows the body 716 comprising the carrier block 432 after the carrier pickup body 450 and the carrier block 432 have been moved to a position adjacent to the thermal management module 440. As shown in FIG27G , the carrier block 432 is supported by at least carrier pick-up arms 451A and 451B. FIG27H illustrates the body 716 of the carrier block 432 after the carrier 432 has been moved by a pick-up device (described herein) and positioned atop the thermal management module 440. As shown in FIG27H , the carrier pick-up arms 451A and 451B are no longer in communication with, e.g., supporting, the edges of the carrier block 432. As shown, the carrier pick-up device and carrier pick-up members can be moved away from the thermal management unit 440, leaving the carrier block 432 and body 716 supported by the thermal management module 440.

[0420] Although not shown, in some embodiments, the lower plate may include two or more substrate tables. In other embodiments, the lower plate may include three or more substrate tables. In still other embodiments, the lower plate may include four or more substrate tables. In other embodiments, the lower plate may include ten or more substrate tables. For example, FIG. 22A illustrates a lower plate having two substrate tables 12 arranged parallel to one another. In embodiments where the lower plate includes two or more substrate tables, in some embodiments, the upper plate will include features complementary to those of the lower plate having the two or more substrate tables (see, for example, FIG. 22B , which includes two cavities 32 adapted to receive at least the substrate 15 of FIG. 22A ). In such embodiments, the complementary upper and lower plates can be brought together to form a chamber suitable for processing two or more substrates together. In some embodiments, the upper and lower plates can be configured such that a divider exists between each substrate table. In this manner, the divider can be used to provide separate processing chambers formed by a single upper and lower plate. In some embodiments, each of the multiple substrates can be heated by the same heating element, or each can be independently heated using a separate heating element, such as a separate heating element embedded within each substrate table. Other features of the chamber are described herein and how the chamber is formed from lower and upper plates having certain complementary features.

[0421] Upper board

[0422] The sample processing assembly of the present disclosure includes at least one upper plate, such as those shown in Figures 9A-9H. Referring to Figure 9A, in some embodiments, upper plate 30 includes a body 33 having an upper engagement surface 31. In some embodiments, upper plate 30 further includes one or more cavities 32 (at least in Figure 9A), such as one or more cavities recessed into body 33. In some embodiments, the upper plate may include a body 33 having an upper engagement surface 31, but unlike the embodiment shown in Figure 9A, the upper plate 30 may not include a recessed cavity (see, for example, Figure 10E).

[0423] In some embodiments, upper plate 30 includes elements that complement features of lower plate 10. In some embodiments, features of upper plate 30 at least partially conform to features present on lower plate 10. In particular, upper plate 30 includes elements that conform to those features of lower plate 10, such that when the upper and lower plates are brought together, a chamber can be formed. In this regard, the upper and lower plates can be considered to be complementary to each other, such that the upper plate is adapted to accommodate features of the lower plate (or vice versa).

[0424] Like the lower plate 10, the upper plate 30 can have any size or shape, as long as the upper plate 30 is complementary to the lower plate 10. For example, as shown in Figures 9A-9H, the upper plate 30 can have a generally rectangular shape, wherein the rectangular upper and lower plates are complementary to each other. Alternatively, as shown in Figures 6A and 6B, the upper plate 30 and lower plate 10 can each have a wedge-based shape, wherein the upper plate 30 includes an upper engagement surface 31 having a size and / or shape that is complementary to the size and / or shape of the lower engagement surface 11 or 711, but wherein the upper and lower surfaces have different configurations.

[0425] The cavity 32 itself can have any size or shape. In some embodiments, the cavity 32 has a size and / or shape suitable for accommodating features of the lower plate 10, such as the substrate stage 12 and any substrate 15 disposed thereon. In this regard, the cavity 32 can have a shape and size complementary to those portions of the lower plate 10 that are raised relative to the lower engagement surface 11 or 711. In some embodiments, the portions of the lower plate 10 that are raised relative to the lower engagement surface 11 can have a first configuration, while the cavity 32 can be adapted to have a second configuration opposite the first configuration (and optionally include additional headspace 40 to accommodate any substrate, fluids disposed thereon, and / or reagents). Furthermore, in some embodiments, the cavity 32 can be adapted to accommodate any fluids, reagents, or samples disposed on the surface of the substrate 15 and / or further accommodate headspace, such as a predetermined dead volume above the substrate surface 15C.

[0426] Figures 9C and 9D provide cross-sectional views of the body 33, illustrating non-limiting examples of the cavity 32. In some embodiments, the dimensions of the cavity 32 are approximately the same as or larger than the dimensions of the substrate 15. In some embodiments, the cavity 32 has a volume at least the same as the volume of the substrate (e.g., the same volume as the substrate plus a predetermined amount of headspace volume). In some embodiments, the cavity 32 is sized to accommodate not only the substrate but also at least a portion of the substrate stage 12. As a non-limiting example, Figure 9C illustrates a cavity 32 having a volume greater than the volume of the substrate 15. For example, the available additional volume can accommodate at least a portion of the substrate stage 12 supporting the substrate 15. By way of another non-limiting example, Figure 9D illustrates a cavity 32 sized to accommodate at least a substrate (e.g., a microscope slide) and any materials disposed on the substrate surface (including fluids, reagents, and / or samples). The cavity further includes a headspace 40 having a predetermined volume. 9C-9F illustrate cavity 32 as having an integral groove, in some embodiments, cavity 32 may be layered, such as shown in FIG10A-10D. In some embodiments, layered cavity 32 may conform to features of lower plate 10, including those that are raised relative to lower engagement surface 11.

[0427] 9B , in some embodiments, the upper plate 30 includes a sealing member 39 that is raised relative to the engagement surface 31, e.g., protruding from the upper engagement surface 31. In some embodiments, the sealing member 39 can be constructed from the same material as the body 33 (e.g., an aluminum body 33 and an aluminum sealing member 39). In some embodiments, the body 33 and the sealing member 39 are machined from a single, integral piece or 3D-printed as a single unit. In some embodiments, the sealing member 39 is adapted to fit within the groove 13 in the lower plate 10 and / or engage the walls of the groove. Although not shown, in other embodiments, the upper plate may alternatively include a groove similar to the groove 13 in the lower plate 10 to accommodate the sealing body, e.g., the upper plate may include a groove having a seal body (such as those described herein) that can engage the sealing body.

[0428] In some embodiments, the upper plate 30 may include one or more heating elements and / or one or more cooling elements, such as one or more elements suitable for heating and / or cooling the material of the upper plate. In some embodiments, the one or more heating and / or cooling elements may be the same as or different from those described for the lower plate (see above). In some embodiments, the active cooling element includes multiple fluid flow channels to facilitate the flow of coolant and thereby provide active cooling of the sample, upper plate, and / or lower plate. In some embodiments, the one or more heating and / or cooling elements are integrated or embedded within the upper plate 30 (see, for example, Figures 9G and 9H). In other embodiments, the one or more heating and / or cooling elements are mounted externally to one or more surfaces of the body 33 of the upper plate 30. In some embodiments, the upper plate 30 may include an integrated heat sink, such as one formed into the sidewalls of the body 33. Additional embodiments illustrating the use of heating and / or cooling elements in the upper and lower plates are shown in Figures 21A-21D.

[0429] In some embodiments, the upper plate 30 may include one or more heating and / or cooling elements 21 (see Figure 29A 29A and 29B ). In some embodiments, the one or more heating and / or cooling elements 21 are embedded within the body 33 of the upper plate 30. For example, FIG. 29A and FIG. 29C each illustrate an upper plate 30 having a body 33, wherein one or more heating and / or cooling elements 21 are shown embedded within the body 33 of the upper plate 30. Any number of heating and / or cooling elements may be embedded within the body 33 of the upper plate 30. Similarly, the body 33 of the upper plate 30 may include any combination of heating and / or cooling elements, for example, 1 heating and / or cooling element; 1 heating element and 1 cooling element; 2 heating elements; 2 cooling elements; 2 heating elements and 1 cooling element; 2 cooling elements and 1 heating element; 2 cooling elements and 2 heating elements; 3 heating elements; 3 cooling elements; 3 heating elements and 1 or 2 cooling elements; 3 cooling elements and 1 or 2 heating elements; 3 cooling elements and 3 heating elements; and so on.

[0430] In some embodiments, the one or more heating and / or cooling elements 21 can have any arrangement within the body 33 of the upper plate 30. For illustrative purposes only, FIG. 29B shows one or more heating elements 21A arranged in parallel with one or more cooling elements 21B. In some embodiments, the one or more heating and / or cooling elements can be arranged side by side (e.g., heating elements 401A and 401B of FIG. 29E ), in a staggered arrangement (e.g., compare elements 401A and 401B with elements 411A and 411B as in FIG. 29F ), can be evenly or randomly spaced, etc. In some embodiments, one or more heating and / or cooling elements can be positioned opposite the cavity 32, with additional heating and / or cooling elements positioned beside the one or more heating and / or cooling elements positioned opposite the cavity 32, with the additional heating and / or cooling elements being located on either side of the one or more heating and / or cooling elements positioned opposite the cavity (see FIG. 29E ). Figure 29D ).

[0431] The body 33 of the upper plate 30 may include one or more bores into which a heating module, such as a heating cartridge, may be inserted. In some embodiments, any number of bores may be provided within the body 33 of the upper plate 30, such as 1, 2, 3, 4, 6, 8, 12, etc. The bores may be arranged in any manner within the body 16 of the lower plate. Thus, the body 16 may accommodate 1, 2, 3, 4, 6, 8, 12, etc. heating cartridges. For example, FIG. 29D and FIG. Figure 29E Each shows a body 33 having two bores 401A and 401B into which the heating cartridges can be inserted.

[0432] In some embodiments, the one or more bores may be evenly spaced from one another. In other embodiments, the one or more bores may be randomly spaced. In some embodiments, the bores open onto the longitudinal sides of the upper plate 30. Alternatively, in other embodiments, the bores may open onto the "short sides" of the lower plate, as shown in FIG29E . The bores themselves may independently have any size and / or diameter.

[0433] In some embodiments, each of the one or more heating cartridges inserted into the body 33 can be independently operated. For example, assuming that the body 33 of the upper plate 30 includes three heating cartridges inserted into three bores, each of the three heating cartridges can be independently operated, such as heating cartridges 1 and 3 can be operated at 80% of their rated power, while cartridge 2 can be operated at 40% of its rated power.

[0434] In other embodiments, the one or more heating and / or cooling elements 21 embedded within the body 33 of the upper plate 30 include one or more fluid channels for circulating a fluid within the body 33 of the upper plate 30. In some embodiments, the fluid is heating oil. In other embodiments, the fluid is a mixture, such as an aqueous mixture including a polyol (e.g., polyethylene glycol or polypropylene glycol) and / or an alcohol (e.g., ethanol or methanol). The one or more fluid channels embedded within the body 33 of the upper plate 30 can have any configuration and can operate independently. For example, and referring to Figures 29D and 29F, the one or more fluid channels 411A and 411B can be arranged as two separate parallel conduits that independently allow the flow of a suitable fluid through the body 33. In this particular example, fluid channels 411A and 411B can each be configured for cooling, each can be configured for heating, or one can be configured for heating and the other for cooling. Likewise, fluid channels 411A and 411B, while both configured for heating, may be configured for heating at different temperatures.

[0435] Alternatively, the one or more fluid channels may include a network of fluid channels 412, such as those shown in FIG29G . The network of fluid channels 412 may include one or more independently controllable inlets 414, outlets 413, or valves (not shown) to direct circulation along a specific fluid flow path or to provide circulation only within a portion of the channel network. In some embodiments, the network of fluid channels may be in communication with a fluid reservoir (not shown), wherein the fluid reservoir may be heated or cooled as desired.

[0436] In some embodiments, any heating and / or cooling elements within the upper plate 30 may operate in conjunction with those heating and / or cooling elements within the lower plate 10 or any thermal management module. As a result, one skilled in the art will appreciate that by controlling the various heating and / or cooling elements present in the upper and lower plates and / or the thermal management module, the temperature of the substrate table, any substrate supported by the substrate table, and / or the chamber may be regulated. For example, the various heating and / or cooling elements present in the upper and lower plates may be operated such that the substrate or sample disposed on the substrate is maintained as the coolest structure within the chamber, e.g., the substrate and / or sample disposed on the substrate is maintained at a lower temperature than the upper plate, lower plate, port, and / or any other structure within the chamber formed by the upper and lower plates. In some embodiments, and with reference to FIG. 40C , the temperature of the cavity 32 of the upper plate 30 is maintained at the same temperature F as the temperature E of any surrounding portion of the body 33 of the upper plate 30. In other embodiments, and with reference to FIG. Figure 40C, the temperature of the cavity 32 of the upper plate 30 is maintained at a temperature F that is lower than the temperature E of any surrounding portion of the body 33 of the upper plate 30. In other embodiments, and referring to FIG40C , the temperature of the cavity 32 of the upper plate 30 is maintained at a temperature F that is higher than the temperature E of any surrounding portion of the body 33 of the upper plate 30.

[0437] In some embodiments, the upper plate may include one or more ports. In some embodiments, the port includes a seal or pressure valve so that gas and / or steam can be released or introduced (such as from a chamber formed by the complementary upper and lower plates). In some embodiments, the one or more ports are configured so that gas and / or steam can be introduced into the chamber formed by the upper and lower plates. In some embodiments, the port is connected to a gas and / or steam source, and the valve can be operated independently so that steam can be introduced, such as into a chamber formed between the upper and lower plates (see Figure 21D). In some embodiments, gas and / or steam are introduced to at least partially heat the substrate and / or sample. In other embodiments, gas and / or steam are introduced to pre-pressurize the chamber formed by the upper and lower plates. In other embodiments, the one or more ports are configured to introduce one or more fluids and / or reagents.

[0438] In some embodiments, the upper plate 30 further includes at least one pressure member. In some embodiments, the at least one pressure member is configured to receive an external force applied to the upper plate 30, the force being applied by one or more force-generating members. In some embodiments, the one or more force-generating members include a motor, a spring, a screw, a lever, a piston (e.g., a mechanically, electrically, pneumatically, or hydraulically actuated piston), a cam mechanism, or any combination thereof. In some embodiments, the externally applied force is in a range from approximately 5 Newtons to approximately 3,000 Newtons. In other embodiments, the externally applied force is in a range from approximately 10 Newtons to approximately 2,000 Newtons. In other embodiments, the externally applied force is in a range from approximately 10 Newtons to approximately 1,000 Newtons. In other embodiments, the externally applied force is in a range from approximately 10 Newtons to approximately 500 Newtons. In still other embodiments, the externally applied force is in a range from approximately 10 Newtons to approximately 250 Newtons. In other embodiments, the externally applied force is in a range from approximately 20 Newtons to approximately 150 Newtons. In some embodiments, the force-generating member applies a predetermined amount of force to the pressure member of the upper plate 30. In some embodiments, the predetermined amount of force applied to the pressure member is less than a predetermined threshold pressure or an amount that does not exceed any predetermined threshold pressure of the internal environment within the chamber formed by the complementary upper and lower plates. In this regard, the force applied by the force-generating member is less than the force that pushes the upper and lower plates apart, and thus, the force-generating member can retract or slide, allowing pressure to be released from the formed chamber.

[0439] In some embodiments, the at least one pressure member is configured to distribute any received force across the body 33 of the upper plate 30, for example, to evenly distribute the received force across the upper plate 30. In some embodiments and as shown in FIG9E , the at least one pressure member comprises two parallel rods 34A and 34B. In some embodiments, the parallel rods 34A and 34B extend along the longitudinal sides 41 of the body 33 of the upper plate 30 (see also FIG11A and FIG11B ). In other embodiments, and with reference to FIG9F , the at least one pressure member comprises a pressure plate 35, for example, an integral pressure plate coupled to the upper surface 33A of the upper plate 30. In some embodiments, the surface area of ​​the pressure plate 35 is greater than the surface area of ​​the upper surface 33A of the upper plate 30. In some embodiments, the surface area of ​​the pressure plate 35 is less than the surface area of ​​the upper surface 33A of the upper plate 30.

[0440] Although not shown, in some embodiments, the upper surface 33A of the body 33 (or for that matter, the pressure plate 35 ) may include one or more mounting points suitable for coupling the upper plate to a support member or subassembly.

[0441] In some embodiments, and as shown in Figures 9G and 9H, the pressure plate 35 is adapted to fit within the void in the body 33. In some embodiments, the pressure plate 35, adapted to fit within the void in the body 33, frictionally engages a raised portion of the body 33 of the upper plate 30. In other embodiments, the pressure plate 35, adapted to fit within the void in the body 33, is fixedly secured to the body 33 of the upper plate 30, for example, by being glued, screwed, or clamped to the upper plate 30. In some embodiments, a heating element 36 is sandwiched between the pressure plate 35 and the body 33. In some embodiments, the pressure plate 35 may also be an integral seal 38.

[0442] In some embodiments, upper plate 30 is formed by machining or milling a solid block of material. In other embodiments, upper plate 30 is formed by a 3D printing process. In other embodiments, upper plate 30 or any portion thereof is produced from a mold. Upper plate 30 may be made of any material. For example, upper plate 30 or any component thereof may be made of a metal or alloy; a ceramic; glass; or a plastic (e.g., derived from a polymer, copolymer, or polymer or copolymer blend). Examples of suitable polymers include, but are not limited to, polyetheretherketone, polyimide, polyetherimide, polytetrafluoroethylene, polysulfone, polyvinylidene fluoride, and polyphenylene sulfide. Examples of suitable metallic materials include aluminum and steel. In some embodiments, if the upper plate is made of metal, the metal may be coated or uncoated (e.g., coated with a fluoropolymer).

[0443] In some embodiments, the upper plate has a total mass ranging from about 30 grams to 200 grams. In other embodiments, the upper plate has a total mass ranging from about 40 grams to 180 grams. In still other embodiments, the upper plate has a total mass ranging from about 40 grams to 160 grams. In still other embodiments, the upper plate has a total mass ranging from about 40 grams to 140 grams. In other embodiments, the upper plate has a total mass ranging from about 40 grams to 120 grams. In other embodiments, the upper plate has a total mass ranging from about 50 grams to 120 grams. In other embodiments, the upper plate has a total mass ranging from about 50 grams to 100 grams.

[0444] In some embodiments, the upper plate has a heat capacity ranging from about 40 J / C to about 110 J / C. In some embodiments, the upper plate has a heat capacity ranging from about 40 J / C to about 100 J / C. In some embodiments, the upper plate has a heat capacity ranging from about 40 J / C to about 90 J / C. In some embodiments, the upper plate has a heat capacity ranging from about 50 J / C to about 80 J / C. In some embodiments, the upper plate has a heat capacity ranging from about 50 J / C to about 70 J / C. In some embodiments, the upper plate has a heat capacity ranging from about 55 J / C to about 75 J / C.

[0445] Additional Sample Processing Kit Parts

[0446] In some embodiments, the sample processing assembly of the present disclosure includes (i) a lower plate; (ii) an upper plate complementary to the lower plate; and (iii) at least one additional component. In some embodiments, the at least one additional component is selected from a subassembly, a support member, a force generating member, a substrate loader, and the like.

[0447] In some embodiments, the sample processing assembly includes one or more subassemblies, such as a track to which one of the lower plate and / or upper plate can be independently coupled (directly or indirectly). In some embodiments, the one or more subassemblies enable the lower plate and / or upper plate to independently move in any one of the x-coordinate, y-coordinate, and z-coordinate directions. For example, and with reference at least to Figures 12A and 12B, in some embodiments, the sample processing assembly of the present disclosure may include a lower plate 10 movably coupled to a subassembly 101. In some embodiments, the subassembly is a track. In some embodiments, the subassembly includes a motor coupled to a carousel or a screw, wherein the carousel or screw can be directly or indirectly coupled to the lower plate 10.

[0448] In some embodiments, subassembly 101 can be linear or curvilinear, such as arcuate, semicircular, etc. In other embodiments, subassembly 101 can have a complex shape, including one or more linear portions and one or more curvilinear portions, such as two linear portions and a central arcuate portion. In some embodiments, two or more lower panels 10 can be coupled to a single subassembly 101, wherein each of the two or more panels 10 can move independently or can be linked so that they move in tandem.

[0449] In some embodiments, the lower plate 10 can be coupled directly to the subassembly 101, such as in tracks along the top and / or sides of the subassembly 101 (see Figures 12A and 12B). In this manner, the lower plate 10 can slide or move directly along the subassembly 101 within the tracks, such as along the plane of the subassembly 101.

[0450] In other embodiments, lower plate 10 can be indirectly coupled to subassembly 101. For example, support member 115A can be coupled to (i) a portion of lower plate 10 and (ii) a track of subassembly 101. In this particular embodiment, support member 115A facilitates movement of lower plate 10 along subassembly 101. In some embodiments, support member 115A is configured such that the plane defined by the upper surface of base 15 remains horizontal, e.g., parallel to the ground, regardless of movement of lower plate 10 along subassembly 101. In some embodiments, support member 115A includes one or more springs.

[0451] In some embodiments, subassembly 101 can be positioned horizontally. This is illustrated in Figure 12A , where subassembly 101 is positioned horizontally within the x,y plane. In this embodiment, horizontal movement of lower plate 10 along the length of subassembly 101 occurs within the x,y plane. Figure 12A also illustrates substrate stage 12 having an upper surface parallel to the ground. Because the track is positioned parallel to the ground, any substrate 15 supported by substrate stage 12 and any sample, fluid, and / or reagent disposed thereon remain parallel to the ground (e.g., within the x,y plane of Figure 12A ) throughout movement of lower plate 10.

[0452] In other embodiments, one end of the subassembly 101 may be elevated along the z-axis relative to the other end. Figure 12B A subassembly 101 is shown having a first end 113 and a second end 114, wherein the first end 113 is elevated relative to the second end 114 along the z-axis (hereinafter referred to as "offset relative to the horizontal" or "horizontally offset"). In such an embodiment, the component of movement of the lower plate 10 along the horizontally offset subassembly 101 will be along the z-axis, e.g., there is a vertical component to the movement. This is further illustrated, for example, in Figures 16A and 16B, which show that the lower plate 10 can move along the subassembly 101, such as from end 114 to end 113, and from a first position to a second position, as well as any intermediate positions therebetween.

[0453] In some embodiments, the horizontal offset angle may be in a range of about 5 degrees to about 70 degrees. In other embodiments, the horizontal offset angle may be in a range of about 5 degrees to about 60 degrees. In still other embodiments, the horizontal offset angle may be in a range of about 5 degrees to about 50 degrees. In still other embodiments, the horizontal offset angle may be in a range of about 10 degrees to about 50 degrees. In still other embodiments, the horizontal offset angle may be in a range of about 15 degrees to about 50 degrees. In other embodiments, the horizontal offset angle may be in a range of about 20 degrees to about 50 degrees. In yet other embodiments, the horizontal offset angle may be in a range of about 20 degrees to about 45 degrees.

[0454] In embodiments where the subassembly 101 is offset relative to the horizontal, the support member 115A may be adapted to orient the lower plate 10, and therefore the upper surface of the substrate table 12, such that any substrate positioned thereon remains parallel to the ground and in the x,y plane. In other embodiments, the lower plate itself is configured such that the lower engagement surface remains parallel to the ground or the surface of the substrate table remains parallel to the ground.

[0455] In some embodiments, the upper plate 30 can be coupled to the subassembly. In some embodiments, and referring to FIG14A , the upper plate 30 can be coupled to the subassembly so that it is fixed in space, such as via support members 115B. In those embodiments where the lower plate 10 is fixed, the upper plate can be movably coupled to the subassembly (movable in any one of the x-, y-, and z-directions). In these embodiments, the upper plate 30 can be coupled to the subassembly in a manner that allows it to move along any one of the x-, y-, and z-axes. Alternatively, the upper plate 30 can be coupled in a manner that allows it to move only within the z-axis.

[0456] In some embodiments, the upper plate may be directly or indirectly coupled to one or more force-generating components. In some embodiments, the one or more force-generating components include a motor, spring, screw, lever, piston (e.g., mechanically, pneumatically, electrically, or hydraulically actuated piston), cam mechanism, or any combination thereof. In some embodiments, the force-generating components facilitate movement of the upper plate along the z-axis (e.g., causing the upper plate to move toward the lower plate) or exert a force along the z-axis (such that a force is exerted to maintain contact between the upper and lower plates, even when the chamber formed by the upper and lower plates is pressurized). In some embodiments, the one or more force-generating components exert a predetermined force on the upper plate. In some embodiments, this predetermined force is limited such that if the pressure within the chamber exceeds a predetermined threshold pressure, the force exerted by the force-generating components is overcome, and the force-generating components will slide or retract to relieve any pressure that may have built up within the chamber above the predetermined threshold pressure. For example, if the one or more force-generating components include a spring or piston, the spring or piston may be configured to exert a predetermined amount of force that is less than any force exerted when the chamber exceeds the predetermined threshold pressure. In other embodiments, a pressure relief port and / or valve is included in either or both of the lower and upper plates so that a predetermined threshold pressure of the chamber is not exceeded.

[0457] In some embodiments, the sample processing assembly may include a substrate loader 302 (see, eg, FIG. 19A ) so that substrates can be moved to or removed from the lower plate 10. Other aspects of the substrate loader and examples of its use in certain sample processing assemblies are further described herein.

[0458] In some embodiments, the sample processing assembly includes one or more base and / or lower plate leveling devices. The base and / or lower plate leveling devices may include, for example, one or more adjustable feet or other adjustment protrusions or springs so that the base can be maintained in a substantially horizontal position during processing, as described herein.

[0459] Chamber formed by upper and lower plates

[0460] As described above, the sample processing assembly of the present disclosure may include one or more chambers. In some embodiments, each chamber is formed by a lower plate and an upper plate, wherein the upper plate is complementary to the lower plate. In some embodiments, the chamber formed by the complementary upper plate 30 and lower plate 10, respectively, is adapted to enclose at least the upper surface 15A of the substrate 15 and any fluids, reagents, and / or samples disposed thereon (see, e.g., FIG. 6B and FIG. 10A-FIG . 10F ). In some embodiments, the complementary upper and lower plates may be configured such that only the substrate or a portion of the substrate is disposed within the formed chamber. In other embodiments, the complementary upper and lower plates may be configured such that the substrate and a portion of the substrate stage are disposed within the formed chamber.

[0461] As described herein, the upper plate 30 and the lower plate 10 are each configured to have a complementary size and / or shape, such that features present on one of the lower or upper plates are accommodated by the other. In some embodiments, when the complementary upper and lower engaging surfaces 31 and 11 (or 711) of the upper and lower plates 30 and 10 (or 710), respectively, contact each other, a chamber may be formed. In some embodiments, the contact between the upper and lower engaging surfaces 31 and 11 (or 711) facilitates the formation of a seal, such as an airtight seal or a moisture-tight seal. In some embodiments, as further described herein, the formation of the seal is further facilitated by applying an external force to one or both of the upper and lower plates.

[0462] In some embodiments, the chambers formed by the lower plate 10 and the upper plate 30, respectively, provide an environment that can be controlled according to user preferences. For example, the chambers can be configured such that the environment within or surrounding the formed chambers differs from the environment outside the chambers, e.g., the environment outside the lower and upper plates. In some embodiments, the formed chambers allow for processing of samples, fluids, and / or reagents disposed on the substrate surface at elevated temperatures (e.g., temperatures greater than room temperature) and / or elevated pressures (e.g., pressures greater than atmospheric pressure), as further described herein.

[0463] In some embodiments, the chamber formed by the complementary upper and lower plates can be internally pressurized, such as by heating the fluid present in the chamber and / or introducing gas and / or steam into the chamber, such as through one or more ports communicated with the formed chamber. For example, the fluid in the reservoir present on the substrate or in the chamber can be heated to increase the pressure in the chamber. In some embodiments, a fluid reservoir is provided in the lower plate or on the substrate itself. In some embodiments, the upper plate itself or a heating element protruding from the upper plate can contact the fluid reservoir (regardless of its position) and heat the fluid in the reservoir to produce steam. The steam produced can then be used to heat and / or pressurize the chamber.

[0464] Alternatively, gas and / or vapor can be introduced into the chamber (such as through one or more ports communicating with the internal environment of the chamber) to facilitate pressurization and / or heating of the chamber. In some embodiments, the pressure within the chamber is regulated by turning one or more heating and / or cooling elements off and on, introducing additional gas and / or vapor into the chamber through one or more ports, releasing gas and / or vapor through one or more ports, or any combination thereof. In some embodiments, the sample processing device and / or chamber includes one or more safety mechanisms to prevent over-pressurization, such as exhaust ports, force-generating members that can apply a force less than the force applied outward from the internal pressurization of the chamber.

[0465] In some embodiments, the chamber is adapted to maintain a temperature in the range of about 70° C. to about 220° C. In other embodiments, the chamber is adapted to maintain a temperature in the range of about 90° C. to about 200° C. In still other embodiments, the chamber is adapted to maintain a temperature in the range of about 95° C. to about 180° C. In other embodiments, the chamber is adapted to maintain a temperature in the range of about 100° C. to about 170° C. In still other embodiments, the chamber is adapted to maintain a temperature in the range of about 110° C. to about 160° C. In still other embodiments, the chamber is adapted to maintain a temperature in the range of about 120° C. to about 150° C.

[0466] In some embodiments, a pressure in the range of about 95 kPa to about 2000 kPa is maintained within the chamber during heating. In other embodiments, a pressure in the range of about 100 kPa to about 1600 kPa is maintained within the chamber during heating. In other embodiments, a pressure in the range of about 150 kPa to about 1050 kPa is maintained within the chamber during heating. In other embodiments, a pressure in the range of about 190 kPa to about 850 kPa is maintained within the chamber during heating. In still other embodiments, a pressure in the range of about 260 kPa to about 750 kPa is maintained within the chamber during heating. In still other embodiments, a pressure in the range of about 300 kPa to about 700 kPa is maintained within the chamber during heating.

[0467] In some embodiments, the force generating member is used to apply a force to the chamber, such as by applying a force to a force generating member on the upper plate through one or more pressure members in communication with the upper plate. In this way, the force applied by the force generating member allows the pressurized internal environment of the chamber to be maintained. As also described above, the force applied by the force generating member can be predetermined. In some embodiments, the predetermined force applied by any force generating member is less than any predetermined threshold pressure. Alternatively, the predetermined force applied by the force generating member is programmed not to exceed any predetermined threshold pressure. In this regard, if the pressure within the chamber exceeds the predetermined threshold pressure, and considering that the predetermined force is set to be below any predetermined threshold pressure or is set to not exceed the predetermined threshold pressure, the force generating member will retract so that the chamber can release or vent the excess pressure, for example, the upper and lower plates can at least partially separate from each other to allow gas and / or vapor to be released to the external environment.

[0468] For example, if the chamber or a substrate, fluid, and / or reagent disposed within the chamber is heated to approximately 160°C and the predetermined threshold pressure is set to 750 kPa, the force-generating member is configured to exert a force less than the predetermined threshold pressure or less than the predetermined threshold pressure. Following this instance, if the pressure exceeds the predetermined threshold pressure of 750 kPa (e.g., if the chamber reaches a pressure of 760 kPa), the force-generating member will retract to allow the release of excess gas and / or vapor. As described herein, the upper and / or lower plates may also include one or more ports and / or valves to allow the release of gas and / or vapor from the chamber when the predetermined threshold pressure is exceeded.

[0469] In some embodiments, the force generating member is configured to retract if the pressure within the chamber exceeds 1250 kPa. In other embodiments, the force generating member is configured to retract if the pressure within the chamber exceeds 1050 kPa. In other embodiments, the force generating member is configured to retract if the pressure within the chamber exceeds 950 kPa. In other embodiments, the force generating member is configured to retract if the pressure within the chamber exceeds 900 kPa. In other embodiments, the force generating member is configured to retract if the pressure within the chamber exceeds 850 kPa. In other embodiments, the force generating member is configured to retract if the pressure within the chamber exceeds 800 kPa. In still other embodiments, the force generating member is configured to retract if the pressure within the chamber exceeds 750 kPa. In still other embodiments, the force generating member is configured to retract if the pressure within the chamber exceeds 700 kPa. In still other embodiments, the force generating member is configured to retract if the pressure within the chamber exceeds 650 kPa. In yet other embodiments, the force generating member is configured to retract if the pressure within the chamber exceeds 600 kPa. In yet other embodiments, the force generating member is configured to retract if the pressure within the chamber exceeds 500 kPa. In still other embodiments, the force generating member is configured to retract if the pressure exceeds 200 kPa, 300 kPa, or 400 kPa. The predetermined threshold pressure at which the force generating member retracts can be set to any value that prevents the chamber pressure from exceeding a value that is deemed unsafe. For example, the predetermined threshold pressure may vary depending on the material of construction of the chamber, local safety regulations, or a combination thereof.

[0470] In some embodiments, one of the lower plate and / or the upper plate may further include one or more temperature sensors and / or pressure sensors. In some embodiments, the one or more temperature sensors are platinum resistance thermometers or thermistors. In other embodiments, the one or more temperature sensors are placed below the substrate. In other embodiments, the one or more temperature sensors are placed on the substrate, for example, at one end of the substrate or on the edge of the substrate. In some embodiments, the one or more temperature sensors are placed on a sample disposed on the substrate.

[0471] The complementarity of the shapes and / or sizes of the features of the upper and lower plates and how this complementarity facilitates the formation of a chamber therebetween is further illustrated in Figures 10A-10C. For example, Figure 10A shows an upper plate 30 and a lower plate 10, respectively, that have been brought together and in contact with one another. In some embodiments, the upper plate 30 and the lower plate 10 contact one another at the interface of the lower engagement surface 11 and the upper engagement surface 31, respectively. As further shown in Figure 10A, the cavity 32 within the upper plate 30 is sized to accommodate at least a portion of the substrate 15 and / or substrate stage 12, as well as any sample, fluid, and / or reagent disposed on the upper surface of the substrate 15. In some embodiments, the cavity 32 further includes a predetermined amount of headspace, e.g., space surrounding the substrate 15. In some embodiments, the chamber has a volume in the range of about 14 cm. 3 to about 25cm 3 In some embodiments, the chamber has a volume between about 15 cm 3 to about 24cm 3 In some embodiments, the chamber has a volume ranging from about 16 cm 3 to about 23cm 3 In some embodiments, the chamber has a volume ranging from about 17 cm 3 to about 22cm 3 In some embodiments, the chamber has a volume ranging from about 18 cm 3 to about 21cm 3 In some embodiments, the chamber has a volume ranging from about 19 cm 3 to about 20cm 3 The volume between.

[0472] Similarly, FIG10B shows upper and lower plates 30 and 10, respectively, brought together and in contact with one another, with upper engaging surface 31 contacting lower engaging plate 11 and / or any sealing body (not shown) disposed within groove 13 of lower plate 10. Furthermore, FIG10B shows a cavity 32 within body 33 of upper plate 30, where cavity 32 is again sized to accommodate at least a portion of substrate 15 and / or substrate stage 12. In some embodiments, cavity 32 of FIG10B includes additional headspace 40 surrounding substrate 15 compared to cavity 32 of FIG10A.

[0473] FIG10C further illustrates that the body 16 of the lower plate 10 can be in communication with a heating element 21, such as any of the types of heating elements described herein. The embodiment shown in FIG10A-10C can also include one or more pressure members in communication with the upper plate 30 (e.g., to receive an externally applied force and impart that force to the upper and / or lower plates), one or more passive and / or active cooling elements (e.g., a heat sink 39 in communication with the lower plate 10 for passive heat removal; a cooling channel in thermal communication with one of the upper and / or lower plates for active heat removal), and / or one or more heating elements (e.g., one or more heating elements embedded in or in communication with the upper and / or lower plates 30, 10 to heat the upper and lower plates or any material disposed on the substrate surface when positioned within the formed chamber) (see, e.g., FIG10D ).

[0474] In some embodiments, and referring to FIG10E , a chamber can be formed by complementary upper and lower plates 30 and 10, respectively, wherein lower plate 10 includes a lower engaging surface 11 and a substrate stage 12 recessed relative to lower engaging surface 11, and upper plate 30 includes an upper engaging surface 31 but does not include a cavity. FIG10F illustrates an alternative embodiment in which upper plate 30 includes a cavity recessed relative to upper engaging surface 31 and lower plate 10 includes a substrate stage 12 recessed relative to lower engaging surface 11. When the two plates are brought into contact with each other, a chamber 32 is formed.

[0475] FIG11A illustrates a non-limiting embodiment, which shows the positioning of the lower plate 10 and the upper plate 30 relative to each other so that complementary features of each of the lower and upper plates can be aligned. In some embodiments, the lower plate 10 is positioned so that at least a portion of the lower engaging surface 11 of the lower plate 10 is aligned with a portion of the upper engaging surface 31 of the upper plate 30. FIG11B depicts the lower plate 10 and the upper plate 30 in physical communication with each other, e.g., at least a portion of the lower engaging surface 11 of the lower plate 10 is in contact with a portion of the upper engaging surface 31 of the upper plate 30. This forms a chamber that encloses at least a portion of the substrate 15 and any sample, fluid, and / or reagent disposed thereon.

[0476] In some embodiments, the chamber formed by the upper plate 30 and lower plate 10, respectively, communicates with one or more heating and / or cooling elements, allowing heating and / or cooling of any substrate or sample positioned with the chamber; or allowing any fluid present within the chamber (such as on the substrate or in a separate reservoir) to be heated to increase the pressure within the chamber. In some embodiments, at least one of the upper plate 30 or lower plate 10 includes a heating element. In some embodiments, at least one of the upper plate 30 or lower plate 10 includes a cooling element (e.g., a passive cooling element or an active cooling element). In some embodiments, the cooling element is an active cooling element utilizing a liquid heat transfer medium. In other embodiments, the cooling element is a passive cooling element, such as a heat sink. In some embodiments, the cooling element is a heat sink coupled to the body of the lower plate. In some embodiments, the cooling element includes a liquid heat transfer medium and a pump or other device for circulating the liquid heat transfer medium (see, e.g., FIG. 21C ). In some embodiments, the cooling element and the heating element operate together to provide a uniform or homogeneous temperature across the substrate and / or sample (e.g., each can be turned off or on as needed and in a controlled manner to provide a substrate or sample with a uniform temperature distribution). In some embodiments, it is believed that a uniform temperature distribution in the sample can be obtained when using a heating surface below the substrate or by introducing an additional heating source above the sample (see Figures 21A and Figure 21B ). In some embodiments, the chamber includes one or more pressure and / or temperature sensors.

[0477] Figures 30A through 30D illustrate alternative embodiments of a chamber formed by an upper plate 30 and a lower plate 10, respectively, and further illustrate the relative positioning of one or more heating and / or cooling elements 21 within the upper and lower plates, as well as the relative positioning of the heating and / or cooling elements to the chamber and / or a substrate disposed within the chamber. For example, Figure 30A illustrates that the upper plate 30 may include one or more heating and / or cooling elements 21 embedded within the body 33 of the upper plate, while also including one or more heating and / or cooling elements 21 embedded within the body 16 of the lower plate.

[0478] FIG30B illustrates an embodiment in which the upper and lower plates each include fluid flow channels (411A-411D), where the fluid flow channels are arranged below the recess 32 and below the substrate stage 12. Each of the fluid flow channels 411A-411D can be independently operated. For example, the type of fluid, flow rate, and temperature of the fluid can be independently controlled for each of the fluid flow channels 411A-411D. The heating cartridges inserted into the bores 401A and 401B of the lower plate 10 can also be independently controlled and used in conjunction with the fluid flow channels to regulate the temperature of the substrate stage, substrate, or any sample disposed thereon. In some embodiments, heating and / or cooling elements can be independently operated to maintain a predetermined temperature gradient between different portions of the upper and lower plates and the substrate stage.

[0479] FIG30C provides another alternative embodiment of a chamber formed by an upper plate and a lower plate, wherein the upper plate and the lower plate include a plurality of fluid flow channels 411A to 411F. FIG30D illustrates an embodiment in which the lower plate 16 is similar to that shown in FIG30B , but in which the upper plate includes a thermoelectric module 402 to enable heating and / or cooling of the body 33 of the upper plate 30. In some embodiments, as shown in FIG30E and FIG30F , the fluid channels of the upper plate 30 and the lower plate 10 can be connected, respectively, so that fluid flows through the fluid channels of one of the upper plate or the lower plate and then flows through the fluid channels of the other of the lower plate or the upper plate.

[0480] In some embodiments, any of the heating and / or cooling elements within the upper plate 30 may operate in conjunction with those within the lower plate 10 or those embedded within one or more thermal management modules. As a result, by independently controlling the various heating and / or cooling elements present in the upper and lower plates, the temperature of the substrate table, any portion of the substrate supported by the substrate table, and / or the chamber may be controlled.

[0481] For example, the various heating and / or cooling elements present in the upper and lower plates can be independently operated so that at least a portion of the substrate or a sample disposed on the substrate is maintained as the coolest structure within the chamber, e.g., the substrate or the sample disposed on the substrate is maintained at a lower temperature than the upper plate, the lower plate, ports, valves, and / or any other structures within the chamber formed by the upper and lower plates. In some embodiments, the heating and / or cooling elements are each independently operated to maintain a predetermined temperature gradient between different portions of the upper and lower plates and the substrate stage.

[0482] Referring to FIG. 40B , different temperature zones can be established, such as temperature zones A, B, C, D, and E. In some embodiments, each of these different temperature zones is adjacent to and / or thermally connected to a portion of the body 16 of the lower plate 10 and / or the body 33 of the upper plate 30. The temperature within each of these zones can be established, maintained, and / or adjusted by independently controlling any number of heating and / or cooling elements in thermal communication with the zone, including heating and / or cooling elements disposed within either the lower plate, the upper plate, or any thermal management module. In some embodiments, at least one of a portion of the substrate 15, the surface of the substrate table 12C, or the substrate table itself is maintained at a lower temperature than other portions of the body 16 of the lower plate 10 or other portions of the body 33 of the upper plate 30. In some embodiments, at least a portion of the substrate disposed within a chamber formed by the upper and lower plates is maintained as the coolest component within the chamber, for example, during a demasking operation (see FIG. 43B - 43C). Figure 43G ).

[0483] For example, and referring again to FIG. 40B , band A can be established at a lower temperature than any of bands B, C, D, and / or E. In some embodiments, bands A and B are maintained at approximately the same temperature, while bands C, D, and / or E are maintained at a relatively lower temperature. Those skilled in the art will appreciate that one or more heating and / or cooling elements positioned near band B will allow bands A and B to remain within a first temperature range, while one or more heating and / or cooling elements positioned near each of bands C, D, and E will allow bands C, D, and E to remain within a second temperature range. In some embodiments, the first temperature range is less than the second temperature range. In some embodiments, the first temperature range is approximately 2% lower than the second temperature range. In other embodiments, the first temperature range is approximately 3% lower than the second temperature range. In some embodiments, the first temperature range is approximately 4% lower than the second temperature range. In some embodiments, the first temperature range is approximately 5% lower than the second temperature range. In some embodiments, the first temperature range is approximately 7% lower than the second temperature range. In some embodiments, the first temperature range is approximately 8% lower than the second temperature range. In some embodiments, the first temperature range is approximately 10% lower than the second temperature range. In some embodiments, the first temperature range is approximately 12% lower than the second temperature range. In some embodiments, the first temperature range is about 15% lower than the second temperature range. In some embodiments, the first temperature range is about 20% lower than the second temperature range. In some embodiments, the first temperature range is about 25% lower than the second temperature range. In some embodiments, the first temperature range is about 30% lower than the second temperature range.

[0484] Figures 41A, 41B, and 41C each illustrate a substrate 15 in thermal communication with one or more heating and / or cooling elements (see, for example, heating and / or cooling elements 401A, 401B, 411A, and 411B in Figure 41B). Figure 41A 41B and 41C illustrate that separate heating and / or cooling elements can be independently operated such that at least a portion of the substrate (or a sample disposed on the substrate) within the chamber formed by the upper plate 30 and the lower plate 10, respectively, can be at a lower temperature than other components disposed within the chamber. In this regard, and as described above, during any temperature ramp-down operation, any condensation that forms within the chamber can be driven to the coolest portion of the chamber, such as the sample or substrate. In this manner, evaporated fluid and / or reagent can be returned to the substrate and / or sample disposed thereon.

[0485] Sample processing

[0486] The sample processing assembly of the present disclosure is configured to facilitate processing of a sample disposed on a substrate surface. In some embodiments, processing of the sample includes unmasking the sample, such as antigen retrieval and / or target retrieval.

[0487] In some embodiments, different sample processing operations can occur in the same area or in different areas of the sample processing assembly. In some embodiments, each of these different sample processing areas can be a pre-designated area, such as an area set next to the sample processing assembly for performing a specific processing operation. Figures 13A and 13B provide two non-limiting examples of pre-designated sample processing areas in the context of a movable lower plate coupled to subassembly 101. As further described below, the lower plate can be moved along subassembly 101 to different pre-designated processing areas. For another example, one or more dispensing devices can be positioned above one or more substrate trays or disc conveyors, such as Figure 20A 20B. In this example, the area defined by the one or more substrate trays, carousel, and dispensing device is a pre-established sample preparation area.

[0488] Alternatively, the sample processing area can be defined by the presence or absence of one or more sample processing assembly components. For example, for a sample processing assembly that includes a fixed lower plate, a user can position a sample-carrying substrate on the upper surface of the substrate table of the fixed lower plate, and this will define the loading area. One or more dispensing devices, mixing devices, and / or liquid removal devices can then be moved to the fixed lower plate to dispense one or more fluids and / or reagents onto the substrate. The presence of one or more dispensing devices in this area defines the area as the sample preparation area.

[0489] Subsequently, the upper plate (and / or force-generating member) can then be moved to the fixed lower plate (after the one or more dispensing devices, mixing devices, and / or liquid removal devices are moved out of the area), so that a chamber can be formed between the movable upper plate and the fixed lower plate. The formed chamber will then define the unmasking area. In this example, any one of the areas can be used as the preparation area and the unmasking area, providing a variety of uses, depending on what devices and / or components are positioned in the area.

[0490] For another example, the lower plate can be moved from a first position to a second position. In some embodiments, the first position is in the loading area and therefore constitutes a pre-specified area. In some embodiments, the second position can be defined as a preparation area or a demasking area, depending on what components are present in the lower plate. For example, one or more dispensing devices can be moved to the lower plate in the second position, thereby defining a preparation area. After one or more fluids and / or reagents are dispensed to the sample-carrying substrate, the upper plate can then be moved to the lower plate in the second position, thereby forming a chamber. In some embodiments, the chamber formed defines a demasking area.

[0491] When the sample processing assembly 100 includes a lower plate 10 movably coupled to the subassembly 101, the lower plate 10 can be moved from a first position to a second position, a third position, and an nth position. For example, in some embodiments, the lower plate 10 can be moved between a loading area, a preparation area, and a demasking area. Of course, the lower plate 10 can be moved to or held in any intermediate area between any loading, preparation, or demasking area. In some embodiments, sample-carrying substrates are loaded onto the lower plate in the loading area. Alternatively, the sample-carrying substrates are loaded onto an intermediate member that is used to hold and / or transport the substrates within the loading area.

[0492] Examples of intermediate components such as substrate loaders 302, substrate holders 310, and / or substrate trays are shown in FIG. Figure 19A - Figure 19C and Figure 20A- Figure 20B. In these embodiments, the intermediate member can transfer the substrate to the lower plate for further processing. After loading the sample-carrying substrate, the sample can be treated with one or more fluids and / or reagents in the preparation area. The demasking operation can then be performed at elevated temperature and / or pressure in the demasking area (see Figure 43A - Figure 43F). For example, once the sample-carrying substrate 15 is moved into position, such as within the chamber formed by the upper plate 30 and the lower plate 10, the demasking operation can allow surface antigens and / or nucleic acids to become clearly distinguishable, regardless of whether a subsequent staining procedure is applied thereto. Demasking can be performed according to any of the methods described in PCT Publication No. WO / 2013 / 079606, the disclosure of which is incorporated herein by reference in its entirety.

[0493] 13A and 13B illustrate various sample processing areas in the case of a movable lower plate. In these embodiments, the lower plate 10 can be moved between the loading area 110, the preparation area 111, and the demasking area 112. In some embodiments, the lower plate 10 can be moved or held at any intermediate area between any of the loading area 110, the processing area 111, or the demasking area 112.

[0494] In some embodiments, the lower plate 10 is initially moved to a loading area, such as shown in FIG13A and FIG13B , if it is not already in such an area. Once in the loading area, a sample-carrying substrate 15 can be positioned on the upper surface 12C of the substrate stage 12 of the lower plate 10, such as by an operator of the sample processing assembly or a system including the sample processing assembly. In some embodiments, the sample-carrying substrate 15 is a microscope slide.

[0495] Next, the lower plate 10 may be moved from the loading area 110 to one of the preparation area 111 or the demasking area 112. In one embodiment, as Figure 13AAs shown, the lower plate 10 moves from the loading area to the preparation area so that one or more fluids and / or reagents can be deposited or dispensed onto the surface of the substrate 15 and / or any sample disposed thereon. In some embodiments, while in the preparation area, one or more demasking agents can be dispensed onto the sample, and demasking can be initiated upon dispensing such agents. In some embodiments, the total volume of the one or more fluids and / or reagents dispensed onto the substrate or the sample disposed thereon ranges from approximately 100 μL to approximately 2000 μL. In some embodiments, the total volume of the one or more fluids and / or reagents dispensed onto the substrate or the sample disposed thereon ranges from approximately 100 μL to approximately 1500 μL. In some embodiments, the total volume of the one or more fluids and / or reagents dispensed onto the substrate or the sample disposed thereon ranges from approximately 100 μL to approximately 1250 μL. In some embodiments, the total volume of the one or more fluids and / or reagents dispensed onto the substrate or the sample disposed thereon ranges from approximately 100 μL to approximately 1000 μL. In some embodiments, the total volume of one or more fluids and / or reagents dispensed onto a substrate or a sample disposed on a substrate is in a range of about 100 μL to about 900 μL. In some embodiments, the total volume of one or more fluids and / or reagents dispensed onto a substrate or a sample disposed on a substrate is in a range of about 150 μL to about 800 μL. In some embodiments, the total volume of one or more fluids and / or reagents dispensed onto a substrate or a sample disposed on a substrate is in a range of about 200 μL to about 750 μL. In some embodiments, the total volume of one or more fluids and / or reagents dispensed onto a substrate or a sample disposed on a substrate is in a range of about 250 μL to about 700 μL. In some embodiments, the total volume of one or more fluids and / or reagents dispensed onto a substrate or a sample disposed on a substrate is in a range of about 250 μL to about 650 μL. In some embodiments, the total volume of one or more fluids and / or reagents dispensed onto a substrate or a sample disposed on a substrate is in a range of about 250 μL to about 600 μL. In some embodiments, the total volume of one or more fluids and / or reagents dispensed onto a substrate or a sample disposed on a substrate is in a range of about 250 μL to about 550 μL. In some embodiments, the total volume of one or more fluids and / or reagents dispensed onto a substrate or a sample disposed on a substrate is in a range of about 300 μL to about 550 μL. In some embodiments, the total volume of one or more fluids and / or reagents dispensed onto a substrate or a sample disposed on a substrate is in a range of about 350 μL to about 550 μL.In some embodiments, the total volume of one or more fluids and / or reagents dispensed to a substrate or a sample disposed on a substrate is about 500 μL.

[0496] Referring again to FIG. 13A , once all fluids and / or reagents have been dispensed onto the sample-carrying substrate, the lower plate 10 can be moved to the demasking area 112. Once in the demasking area 112, the lower plate 10 and / or upper plate 30 can be independently moved so that at least a portion of the lower engaging surface 11 contacts a portion of the upper engaging surface 31 to form a chamber that at least partially encloses the sample-carrying substrate 15 and any fluids and / or reagents disposed thereon. In some embodiments, the chamber facilitates processing samples, fluids, and / or reagents in a controlled environment and in a predetermined manner. In some embodiments, the controlled environment allows for processing samples at elevated temperatures and / or pressures compared to the environment outside the chamber. In some embodiments, the demasking operation is performed within a sealed chamber (see, for example, FIG. 43A through FIG. 43F ).

[0497] In some embodiments, one or more heating elements can be activated to heat the fluid present within the chamber, thereby increasing the pressure within the chamber (which concomitantly increases the pressure applied to any sample disposed on the substrate). In some e...

Claims

1. A system comprising: (i) at least one sample processing assembly comprising: (1) a lower plate comprising a body including (a) a substrate stage having an upper surface adapted to horizontally support a substrate, (b) a lower engagement surface, and (c) a first lower temperature adjustment element in thermal communication with the substrate stage, the lower plate further comprising a second lower temperature adjustment element and a third lower temperature adjustment element, each temperature adjustment element being independently operable; and (2) an upper plate comprising an upper engagement surface complementary to the lower engagement surface, the upper plate further comprising a cavity recessed relative to the upper engagement surface; and (ii) Staining module.

2. The system of claim 1, wherein the first lower temperature regulating element is positioned below the substrate stage.

3. The system of any one of claims 1-2, wherein the second lower temperature regulating element and the third lower temperature regulating element are each positioned adjacent to the first lower temperature regulating element.

4. The system of claim 3, wherein the first lower temperature regulating element has a first heat output, the second lower temperature regulating element has a second heat output, and the third lower temperature regulating element has a third heat output, wherein the first heat output is less than either the second heat output or the third heat output.

5. The system of claim 4, wherein a thermal gradient is maintained between the first lower temperature regulating element and the second lower temperature regulating element and between the first lower temperature regulating element and the third lower temperature regulating element.

6. The system of claim 5, wherein the thermal gradient maintained between the first lower temperature regulating element and the second lower temperature regulating element is in the range of 2°C to about 10°C; and wherein the thermal gradient maintained between the first lower temperature regulating element and the third lower temperature regulating element is in the range of 2°C to about 10°C.

7. The system of any one of claims 1 to 2, wherein the lower plate is modular.

8. The system of claim 7, wherein a modular lower panel is transportable to the upper panel.

9. The system of claim 8, wherein the upper plate is coupled to a force generating member.

10. The system of claim 9, wherein the force generating member is configured to retract if a pressure within a chamber formed by the upper plate and the lower plate exceeds a predetermined threshold.

11. The system of any one of claims 1 to 2, wherein the first lower temperature regulating element in thermal communication with the substrate table is configured to maintain the substrate table or a substrate disposed thereon as the coolest component within a chamber formed by the upper plate and the lower plate.

12. The system of any one of claims 1 to 2, further comprising at least one substrate transfer device.

13. The system of claim 12, wherein the at least one substrate transfer device is selected from the group consisting of a gripper device, a forklift device, and a carrier transport device.

14. The system of any one of claims 1 to 2, further comprising one or more substrate loading stations.

15. Use of the system according to any one of claims 1 to 14 in preparing a demasked sample.

16. Use of the system according to claim 15, wherein the sample is a histological sample.

17. Use of the system according to claim 15, wherein the sample is a cytology sample.

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