SYSTEM WITH A TREATMENT CHAMBER FOR BIOLOGICAL SAMPLES AND USE

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present disclosure claims the benefit of the filing date of U.S. Provisional Application No: 62 / 891,118 filed on August 23, 2019, and the benefit of U.S. Provisional Application No. 62 / 847,388 filed on May 14, 2019.FIELD OF THE DISCLOSURE

[0002] The present disclosure relates to a system for unmasking protein antigens and nucleic acid targets from fixed biological samples.BACKGROUND OF THE DISCLOSURE

[0003] Fixation of tissue and cell samples is used to help ensure that the morphology of the sample and the spatial distribution of biomolecules is preserved and thus enable a diagnosis by a pathologist. As an adjunct to morphological indicators of disease, the presence of particular proteins and / or nucleic acid sequences can be used to further characterize a disease state and, in some instances, is used by an oncologist to direct therapy of the disease. However, fixation can also interfere with the detection of proteins and nucleic acids in a sample. As such, for immunohistochemical (IHC) procedures and in-situ hybridization (ISH) procedures, an unmasking step (also known as "antigen retrieval" or "target retrieval" for IHC and ISH, respectively) is often used make protein antigens or nucleic acid targets accessible to detection reagents such as antibodies or probes.

[0004] Fixation of samples is routinely accomplished using neutral-buffered formalin (NBF). It is believed that the formaldehyde in NBF preserves tissue and cell morphology by forming cross-links between reactive groups on proteins and nucleic acids in the sample, and these cross-links can lead to certain portions of the molecules being rendered undetectable. For example, formaldehyde preserves or fixes tissue or cells predominantly by cross-linking primary amine groups in proteins with other nearby nitrogen atoms in protein or DNA through a -CH 2 - linkage. The process of tissue fixation however, frequently masks antigens on specific proteins for which detection is desirable for diagnostic and prognostic purposes. A variety of methods are used to reverse the effects of formalin fixation and provide access to antigens and targets in fixed biological samples. None of these methods, however, are believed to be completely successful in all instances, and some are more amenable to automation that others. US 9 945 763 B1 describes heat induced antigen retrieval systems for biological specimens which may include a sealable heating pressure chamber, a programmable process controller, a nonpareil operating element, and perhaps even a substantially user-disencumbering autonomous processing component of a plurality of biological samples perhaps using various user selected protocols, and the like. US 2018 / 017471 A1 describes an automated in situ heat induced antigen recovery and staining method and apparatus for treating a plurality of microscope slides. The process of heat induced antigen recovery and the process of staining the biological sample on the microscope slide are conducted in the same apparatus, wherein the microscope slides do not need to be physically removed from one apparatus to another. The reaction conditions for treating a slide can preferably be controlled independently, including the individualized application of reagents to each slide and the individualized treatment of each slide. EP 3 427 829 A1 describes a device and method for in situ temperature-induced antigen retrieval of samples wherein all steps are performed under a pressure higher than the atmospheric pressure on a sample immobilized on a sample support which can be further subjected to staining and imaging on the same sample support, optionally by cycle multiplexing that enables imaging of various molecular targets through multi-molecular read-outs on the same sample in a rapid, highly sensitive and reliable manner. WO 2018 / 073283 A1 describes a system and a method for treatment of biological samples. An automated biological sample staining system comprises at least one microfluidic reagent applicator; at least one bulk fluid applicator; at least one fluid aspirator; at least one sample substrate holder; at least one relative motion system; and a control system that is programmed to execute at least one staining protocol on a sample mounted on a substrate that is held in the at least one sample substrate holder. US 2010 / 068757 A1 describes an automated microscope slide staining system and staining apparatus and method that features a plurality of individually operable miniaturized pressurizable reaction compartments or a pressurizable common chamber for individually and independently processing a plurality of microscope slides. The apparatus preferably features independently movable slide support elements each having an individually operable heating element.BRIEF SUMMARY OF THE DISCLOSURE

[0005] The invention is set out by the pending claims. The examples are not scope of the claims but are for reference only. The present disclosure is directed to specimen processing assemblies including (a) a lower plate, and (b) an upper plate which is complementary to the lower plate. Upper and lower plates which are "complementary" each include features or sets of features which complement one another. For example, a lower plate may have a first set of features (e.g. a substrate stage and a lower engagement surface) and an upper plate may have a second set of features (e.g. a cavity and an upper engagement surface), whereby the first and second set of features are complementary to each other. In some examples, the specimen processing assemblies include complementary lower and upper plates which are independently movable. For example, the lower plate may be coupled to a sub-assembly which is movable toward an upper plate (e.g. a fixed upper plate or a movable upper plate). By way of further example, the upper plate may be coupled to a sub-assembly which is movable toward a lower plate (e.g. a fixed lower plate or a movable lower plate). In some examples, the lower plate has a modular design ("modular lower plate") which allows it to be used in a multitude of different specimen processing assemblies.

[0006] In some examples, the specimen processing assemblies include a chamber formed from the complementary upper and lower plates (or a modular lower plate having features complementary to an upper plate). In some examples, the specimen processing assembly is adapted for unmasking, e.g. antigen retrieval and / or target retrieval, a specimen disposed on a substrate and provided within the chamber. In some examples, a specimen disposed on a substrate and / or at least a portion of the substrate itself is maintained at a temperature which is less than any other component within the chamber during an unmasking operation. For example, example the specimen disposed on the substrate or the portion of the substrate itself may be maintained as the "coldest" component within the chamber during an unmasking operation. Other aspects of specimen processing assemblies and the components of such specimen processing assemblies are described further herein. The present disclosure is also directed to systems including one or more independently operable specimen processing assemblies.

[0007] In one aspect of the present disclosure is a specimen processing assembly including (a) a lower plate, and (b) an upper plate which is complementary to the lower plate. In some examples, the lower and upper plates have complementary polygonal shapes. In some examples, the lower and upper plates have complementary wedge-based shapes. In some examples, at least one of the lower and upper plates are movable. In some examples, both of the lower and upper plates are independently movable.

[0008] In some examples, the lower plate includes a first set of features and the upper plate includes a second set of features, wherein the first and second sets of features are complementary to each other. In some examples, the first set of features includes a lower engagement surface and one or more substrate stages. In some examples, the one or more substrate stages are raised relative to at least a portion of the lower engagement surface. In some examples, the one or more substrate stages are recessed relative to at least a portion of the lower engagement surface. In some examples, the second set of features include an upper engagement surface. In some examples, the second set of features include an upper engagement surface and one or more cavities. In some examples, the one or more cavities are recessed relative to at least a portion of the upper engagement surface. In some examples, each of the lower and upper plates include additional features including one or more ports, one or more heating elements, one or more cooling elements, one or more substrate alignment members, etc. In some examples, the one or more ports may intake ports through which one or more gases and / or steam may be supplied. In some examples, the one or more ports may be vent ports through which gas and / or steam may be released.

[0009] In some examples, the lower plate has a modular design. In some examples, a modular lower plate includes a body having a lower engagement surface. In some examples, the modular lower plate includes a thermal management module. In some examples, the body may rest on, engage with, or couple to the thermal management module. In some examples, the body includes a substrate stage that is part of and integral with body. In some examples, the body may be picked up, transported to, and deposited onto a thermal management module while a substrate is supported by the substrate stage of body (where the body including the substrate stage and the thermal management module together constitute the modular lower plate). In some examples, the body is included within a carrier block. In some examples, the carrier block and the body may be picked up, transported to, and deposited onto a thermal management module. In other examples, the body includes a lower engagement surface but does not include an integral substrate stage. Rather, the modular lower plate includes a body having a separable substrate stage. In this way, a separable substrate stage may be used as a carrier for a substrate. For example, a substrate may be disposed onto the separable substrate stage and together the substrate and the separable substrate stage pair may be picked up, transported to, and deposited onto a body. In some examples, a substrate and a separate substrate stage pair remain together throughout all or at least some of the processing steps to which the specimen disposed on the substrate may be subjected, for example, through all or part of the steps used to prepare a sample for microscopic analysis, such as from baking through coverslipping or from baking through staining. In some examples, a user places a substrate bearing a sample onto the separate substrate stage and inputs the substrate / substrate stage pair into a system and following processing of the sample, retrieves the substrate / substrate stage from the system, and then removes the substrate with a processed sample thereon for analysis.

[0010] In some examples, the complementary upper and lower plates include complementary upper and lower engagement surfaces, respectively. In some examples, the complementary upper and lower engagement surfaces both include complementary flat surfaces. In some examples, the complementary upper and lower engagement surfaces both include complex complementary surfaces, such as complementary surfaces including curvilinear or arcuate shapes. In some examples, the complementary upper and lower engagement surfaces are configured such that a sealing engagement may be formed when the upper and lower engagement surfaces at least partially contact one another or contact a seal body disposed therebetween. In some examples, a sealing engagement between the complementary upper and lower engagement surfaces facilitates the formation of a sealed chamber.

[0011] In some examples, the sealed chamber is heated and / or pressurized for a predetermined amount of time and then cooled. In some examples, the sealed chamber is heated and / or pressurized for a predetermined amount of time and then opened without first cooling down. In some examples, at least a portion of the specimen disposed on the substrate and / or at least a portion of the substrate itself is maintained at a temperature which is lower than any other component within the sealed chamber during an unmasking operation. For example, the specimen disposed on the substrate or the substrate itself may be maintained as the "coldest" component within the sealed chamber during all steps of the unmasking operation, such as during heating, pressurization, cooling, depressurization, quenching, the dispensing of additional fluids, etc. For example, the specimen and / or the portion of the substrate may be maintained at a temperature which is at least about 2°C less than any other component within the sealed chamber. By way of another example, the specimen and / or the portion of the substrate may be maintained at a temperature which is at least about 5°C less than any other component within the sealed chamber.

[0012] In some examples, any fluids and / or reagents needed for performing an unmasking operation are first introduced to a specimen disposed on the substrate, to a portion of the substrate itself, and / or to a reservoir, and then the chamber is sealed. By way of example, a total volume of between about 250µL to about 1000µL of one or more fluids and / or reagents may dispensed to a specimen disposed on a substrate, a portion of the substrate itself, and / or a reservoir within the lower plate before the formation of a sealed chamber between the upper and lower plates. In some examples, no additional fluids and / or reagents are applied after the formation of a sealed chamber.

[0013] In some examples, one or more additional fluids and / or reagents are dispensed to the specimen and / or the substrate in the sealed chamber as part of the unmasking operation. For example, between about 250µL to about 1000µL of one or more additional fluids and / or reagents may dispensed into the sealed chamber as part of the unmasking operation. In some examples, one or more additional fluids and / or reagents are dispensed into the sealed chamber as part of the unmasking operation such that at least about 90% of the volume of the chamber is filled with fluids and / or reagents. In some examples, one or more additional fluids and / or reagents are dispensed into the sealed chamber as part of the unmasking operation such that at least about 95% of the volume of the chamber is filled with fluids and / or reagents. In some examples, one or more additional fluids and / or reagents are dispensed into the sealed chamber as part of the unmasking operation such that at least about 99% of the volume of the chamber is filled with fluids and / or reagents.

[0014] In some examples, either a body of the lower plate or the one or more substrate stages are themselves configured such that an upper surface of the substrate stage is horizontal, for example parallel to the ground. In those examples where the lower plate is movable, the one or more substrate stages and / or the lower body is configured such that as the lower plate is moved, a substrate supported by the upper surface remains horizontal. In some examples, the upper surface of the substrate stage remains in a horizontal orientation throughout the course of the movement of the lower plate, and this is regardless of whether the movement of the lower plate remains entirely parallel to the ground or if there is a vertical component to the movement of the lower plate (including those examples where there is a completely vertical movement). In examples where the bodies of the lower and upper plates have complementary wedge-based shapes, the raised substrate stage may itself have a substantially wedge-based shape such that an upper surface of the raised substrate stage remains horizontal while the lower plate is moved.

[0015] In some examples, the complementary lower and upper plates are independently movable such that they contact one another at an interface of the complementary lower and upper engagement surfaces. In some examples, the movement of any of the upper and lower plates is affected with one or more of a motor, a screw, a lever, a spring, a cam mechanism, a piston, or any combination thereof. In some examples, an additional external force is applied to one or both of the upper and / or lower plates after the plates are brought into at least partial contact with one another. In some examples, the application of the additional external force facilitates a sealing engagement between the upper and lower plates to be maintained (with or without the use of a seal body, as described below). In some examples, the external force is applied with one or more force generating members (e.g. one or more of a motor, a piston, a spring, a screw mechanism, a lever and / or a cam mechanism).

[0016] In some examples, the lower plate and the upper plate are both moved simultaneously toward each other until they both contact each other (for example their respective upper and lower engagement surfaces contact one another), and then one or more force generating members are engaged to apply an additional force on at least the upper plate. In some examples, one or more force generating members in communication with at least the upper plate may be engaged to force the upper plate against the lower plate, thereby further facilitating a sealing engagement between the upper and lower plates, or enabling a sealing engagement to be maintained as the internal pressure in a chamber formed from the upper and lower plates increases (for example, where the internal pressure increases during an unmasking operation and / or from pre-pressurization).

[0017] In some examples, each of the lower and upper plates are independently movable in any of the x-, y-, and z-coordinate directions. In some examples, the lower plate and the upper plate are both simultaneously movable toward each other in any of the x-, y-, and z-coordinate directions. In some examples, one of the upper or lower plates may be moved to a predetermined position and, simultaneously or subsequently, the other of the upper or lower plate may be moved toward the positioned plate. In some examples, the lower plate may be moved to a predetermined position and then, simultaneously or subsequently, the upper plate may be moved toward the positioned lower plate. In some examples, the lower plate is moved to a predetermined position and the upper plate may be moved downward toward the lower plate.

[0018] In some examples, one of the lower or upper plates is movable, while the other of the lower or upper plate is not movable. In some examples, the upper plate is fixed, and the lower plate is movable toward the upper plate. In some examples, the lower plate is fixed, and the upper plate is movable toward the lower plate.

[0019] In some examples, the lower plate moves from a loading area to at least one of a preparation area or an unmasking area. In some examples, the movement of the lower plate facilitates the treatment of a substrate supported by the lower plate with one or more fluids and / or reagents. In some examples, the lower plate is fixed and one or more dispense devices are moved to the fixed lower plate to dispense one or more fluids and / or reagents to a substrate supported by the lower plate. By way of example, a total volume of between about 250µL to about 1000µL of one or more fluids and / or reagents may be dispensed to a specimen disposed on a substrate, a portion of the substrate itself, and / or a reservoir within the lower plate before the formation of a sealed chamber between the lower and upper plates. In some examples, no additional fluids and / or reagents are dispensed to the substrate once the substrate is positioned within the sealed chamber.

[0020] In some examples not forming part of the present invention, the complementary lower and upper engagement surfaces facilitate a sealing engagement without the use of any seal body disposed therebetween. At least one of the lower plate and the upper plate includes at least one seal body. In some examples, the seal body may be disposed within a groove of either the complementary upper and / or lower plates, and the seal body, along with the complementary upper and lower engagement surfaces, facilitates a sealing engagement between the upper and lower plates. In some examples, the at least one seal body is removable. In some examples, a seal body may be disposed between the complementary upper and lower engagement surfaces without being disposed within a groove. In some examples, the seal body is positioned on the surface of the lower engagement surface. In these examples, when an upper plate is brought into contact with the sealing element positioned on the lower engagement surface, a sealing engagement may be maintained, such as by exerting an external force onto the lower plate and / or the upper plate with one or more force generating members. In some examples, the at least one removable seal is integrated within a removable seal attachment, wherein the removable seal attachment is configured to engage a portion of a periphery of the lower plate or the upper plate. In some examples, one of the complementary lower or upper engagement surfaces includes a raised sealing member protruding from a plane formed by the one of the complementary lower or upper engagement surfaces, and where the other of the complementary lower or upper engagement surface includes a channel which is complementary to the raised sealing member.

[0021] In some examples, at least one of the upper and lower plates further includes one or more heating and / or cooling elements. In some examples, the cooling elements are active cooling elements. In some examples, the active cooling elements include a tubes 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 chiller such that a liquid heat transfer medium may be circulated through the tube to effectuate cooling of at least a portion of the lower plate, the upper plate, and / or a substrate disposed on a substrate stage. In some examples, the cooling elements are a passive cooling elements. In some examples, the passive cooling elements are a heat sink. In some examples, only the upper plate includes a heating element. In some examples, at least one of the upper or lower plates includes a heating element, and wherein the lower plate includes a passive or active cooling element. In some examples, only the lower plate includes a heating element.

[0022] In examples where both the upper and lower plates include heating and / or cooling elements, the heating and / or cooling elements may be independently operable, as described herein. In some examples, any of the heating and / or cooling elements within the upper plate may be operated in conjunction with those heating and / or cooling elements within the lower plate, or those heating and / or cooling elements 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 stage, any portion of a substrate supported by the substrate stage, and / or the chamber may be controlled. In some examples, independent control of the various heating and / or cooling elements enable thermal gradients to be formed and maintained, e.g. thermal gradients between a substrate supported by a substrate stage and the walls of the interior of the formed chamber.

[0023] In some examples, at least one of the upper and lower plates further includes a sensor, such as a temperature sensor. In some examples, the upper plate includes a temperature sensor which contacts the substrate and / or the sample disposed on the substrate. In some examples, the lower plate includes a temperature sensor which contacts the substrate. In some examples, the data from the temperature sensor is monitored such that one or more heating and / or cooling elements disposed within the lower and / or upper plates may be controlled. For example, the sensor may provide feedback such that the one or more heating and / or cooling elements disposed within the lower and / or upper plates may be controlled to maintain a specimen or a portion of the substrate as the coldest components within the sealed chamber.

[0024] Another aspect of the present disclosure is a specimen processing assembly including (i) a lower plate coupled to a sub-assembly, wherein the lower plate includes a lower engagement surface and one or more substrate stages, wherein the one or more substrate stages includes an upper surface adapted to hold a substrate horizontally; and (ii) an upper plate having an upper engagement surface complementary to the lower engagement surface. In some examples, the upper plate further includes one or more cavities. In some examples, the one or more cavities each receive at least a portion of the one or more substrate stages. In some examples, one of the lower plate and the one or more substrate stages are configured such that the upper surface of the one or more substrate stages remains horizontal as the lower plate traverses the sub-assembly (for example any substrate supported by the one or more substrate stages remain parallel to the ground throughout the movement of the lower plate along the sub-assembly, and regardless of whether the movement is completely horizontal along the sub-assembly or the movement includes a vertical component to the movement, or even a purely vertical movement). In some examples, the substrate is a microscope slide.

[0025] In some examples, the lower plate is coupled to a sub-assembly which functions to move the lower plate into contact with the upper plate. In some examples, the sub-assembly is moved with sufficient force such that the movement of sub-assembly itself is sufficient to create a seal between the lower plate coupled to the sub-assembly and the upper plate. For example, the sub-assembly can include a means for translating the lower plate along a rail (e.g. a rail arranged horizontally or a rail having one end offset from the horizontal, as described below). In some examples, the sub-assembly includes a motor adapted to move the lower plate along the rail, e.g. from a loading position to an unmasking position.

[0026] In some examples, the sub-assembly is arranged horizontally. In this configuration, the lower plate moves along the horizontally arranged sub-assembly. In some examples, the sub-assembly is arranged such that a first end of the sub-assembly is raised vertically relative to a second end of the sub-assembly, for example the sub-assembly is offset from the horizontal. In this particular embodiment, as the lower plate moves along the sub-assembly it does so with a motion that includes a vertical component, for example as the lower plate is moved along any of the x- and / or y- directions of the horizontally offset sub-assembly, there is a concomitant movement in the z-direction given the horizontal offset of the rail. In some examples, the rail is offset from the horizontal at an angle ranging from between about 5 degrees to about 70 degrees. In other examples, the horizontal offset angle ranges from between about 5 degrees to about 60 degrees. In yet other examples, the horizontal offset angle ranges from between about 10 degrees to about 50 degrees. In further examples, the horizontal offset angle ranges from between about 20 degrees to about 50 degrees. In yet further examples, the horizontal offset angle ranges from between about 20 degrees to about 45 degrees. In some examples, the sub-assembly further includes a motor adapted to move the lower plate along the sub-assembly.

[0027] In some examples, the upper plate is coupled to a support member and the lower plate is moved (either along a horizontal sub-assembly or a sub-assembly which is horizontally offset) to a position where the lower engagement surface contacts the complementary upper engagement surface of the upper plate. In some examples, 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 the complementary upper engagement surface of the upper plate, and where the one or more springs exert a downward force onto the upper plate as the lower plate contacts the upper plate.

[0028] In some examples, the lower plate is moved to a predetermined position underneath the upper plate and the upper plate is moved at least along the z-axis toward the positioned lower plate. In other examples, the lower plate is moved near an upper plate, and the upper plate is moved either simultaneously or subsequently (in any of the x, y, and z directions) toward the lower plate until the upper engagement surface of the upper plate contacts the lower engagement surface of the lower plate. In yet other examples, the lower plate and upper plate are both simultaneously moved toward each other (here, the movement of each of the lower and upper plates may independently be in any of the x-, y-, and / or z- directions). In some examples, the lower plate and the upper plate are both moved simultaneously toward each other until they both contact each other (for example their respective upper and lower engagement surfaces contact one another), and then force generating member in communication with the upper plate is engaged. In some examples, the engagement of the force generating member causes a force to be exerted onto at least the upper plate. In some examples, the force exerted onto the upper plate facilitates a sealing engagement between the upper and lower plates. In some examples, a predetermined amount of force is exerted by the force generating member onto the upper plate. In some examples, the predetermined amount of force exerted by the force generating member onto the upper plate is greater than a force generated through the internal pressurization of a chamber formed from the upper and lower plates but lower than a predetermined threshold force above which unsafe pressures could be generated within the chamber. In some examples, this predetermined force exerted by the force generating member is limited such that should pressure within the chamber exceed a predetermined pressure, the force exerted by the force generating member is overcome and the force generating member will slip or give way to relieve pressures above the predetermined pressure that might develop within the chamber.

[0029] In some examples, the specimen processing assembly includes a chamber. In some examples, the chamber is formed from the complementary lower and upper plates. In some examples, the complementary lower and upper engagement surfaces of the lower and upper plates, respectively, facilitate a sealing engagement between the upper and lower plates such that a chamber is formed therebetween. In some examples, the complementary lower and upper engagement surfaces provide a sealing engagement without the use of any seal body disposed therebetween. In some examples, a seal body may be disposed within a groove of either the upper and / or lower plates, and the seal body, along with the upper and lower engagement surfaces, facilitates a sealing engagement between the upper and lower plates. In some examples, a seal body may be disposed between the upper and lower engagement surfaces without being disposed within a groove, e.g. the seal body lies on the surface of the lower engagement surface. In some examples, one of the lower or upper engagement surfaces includes a raised sealing member protruding from the plane formed by the one of the lower or upper engagement surfaces, and where the other of the lower or upper engagement surface includes a channel which is complementary to the raised sealing member.

[0030] In some examples, one or both of the lower and upper plates include one or more heating and / or cooling elements. In some examples, the one or more heating and / or cooling elements within the lower and upper plates are operated together such that, for example, predetermined thermal gradients are established and maintained within the chamber. For example, the one or more heating and / or cooling elements within the lower and upper plates are operated together such that a thermal gradient is established and maintained between a substrate stage and between other components within the chamber, e.g. the walls defining the interior of the chamber. In some examples, the one or more heating and / or cooling elements are independently controlled such that the specimen disposed on the substrate and / or a portion of the substrate itself is maintained at a temperature which is less than any other component (e.g. chamber walls, ports, valves, sensors, probes, etc.) within the chamber. For example, the specimen and / or the portion of the substrate may be maintained at a temperature which is at least about 2°C less than any other component within the sealed chamber. By way of another example, the specimen and / or the portion of the substrate may be maintained at a temperature which is at least about 5°C less than any other component within the sealed chamber. In some examples, the upper plate further includes one or more ports. In some examples, the one or more ports permit the introduction of one or more gases and / or steam into the chamber formed from the lower and upper plates. For example, steam may be introduced to heat a specimen within the chamber and concomitantly pressurize the chamber. In some examples, the upper plate further includes one or more valves to facilitate the release of gases and / or steam from within the chamber formed from the lower and upper plates. For example, one or more valves may be opened for a predetermined amount of time such that pressure may be released from the sealed chamber. Alternatively, the sealed chamber may be opened immediately after an unmasking operation is completed, e.g. the sealed chamber may be opened without first cooling down or depressurizing.

[0031] In some examples, an unmasking operation conducted within the chamber formed from the complementary lower and upper plates is performed using fluids and / or reagents supplied to the substrate prior to the formation of the chamber. In some examples, no additional fluids and / or reagents are dispensed to the substrate when the substrate is positioned within the chamber and after the chamber is sealed.

[0032] In some examples, the chamber may include one or more ports into which one more additional fluids and / or reagents may be added directly into the sealed chamber, such as part of the unmasking operation. In some examples, one or more additional fluids and / or reagents are dispensed to the specimen and / or the substrate in the sealed chamber as part of the unmasking operation. For example, between about 250µL to about 1000µL of one or more additional fluids and / or reagents may dispensed into the sealed chamber as part of the unmasking operation. In some examples, one or more additional fluids and / or reagents are dispensed into the sealed chamber as part of the unmasking operation such that at least about 90% of the volume of the chamber is filled with fluids and / or reagents. In some examples, the lower plate may include one or more ports, e.g. vacuum ports, for removing excess liquids from within the chamber. In some examples, the vacuum ports in the lower plate may be controlled after substantially filling the chamber with additional fluids and / or reagents.

[0033] Another aspect of the present disclosure is a system including one or more independently operable specimen processing assemblies, one or more dispense 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 specimen processing assemblies and at least the one or more dispense devices. In some examples, each of the one or more independently operable specimen processing assemblies include one or more lower plates and one or more upper plates, where each of the one of more upper plates are complementary to each of the one or more lower plates.

[0034] In some examples, the lower plates have a modular design. In some examples, a modular lower plate includes a body having a lower engagement surface. In some examples, the modular lower plate includes a thermal management module. In some examples, the body may rest on, engage with, or couple to the thermal management module. In some examples, the body includes a substrate stage that is part of and integral with body. In other examples, the body includes a lower engagement surface but does not include an integral substrate stage. Rather, the modular lower plate includes a body having a separable substrate stage. In this way, a separable substrate stage may be used as a carrier for a substrate. For example, a substrate may be disposed onto the separable substrate stage and together the substrate and the separable substrate stage pair may be picked up, transported to, and deposited onto a body. In some examples, a substrate and a separate substrate stage pair remain together throughout all or at least some of the processing steps, for example, through all or part of the steps used to prepare a sample for microscopic analysis, such as from baking through coverslipping or from baking through staining. In some examples, a user places a substrate bearing a sample onto the separate substrate stage and inputs the substrate / substrate stage pair into a system and following processing of the sample, retrieves the substrate / substrate stage from the system, and then removes the substrate with a processed sample thereon for analysis.

[0035] In some examples, the system includes at least two independently operable specimen processing assemblies, and wherein one of the at least two specimen processing assemblies includes a sealed chamber (e.g. a chamber formed from one upper plate and one complementary lower plate or a complementary modular lower plate).

[0036] In some examples, the lower and upper plates include one or more independently operable heating and / or cooling elements. For example, the independently operable heating and / or cooling elements may be present in the lower plate, the upper plate, or both the lower and upper plates. In some examples, the chamber is in communication with at least two heating and / or cooling elements. In some examples, the heating and / or cooling elements within the upper plate may be operated in conjunction with those heating and / or cooling elements 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 a substrate supported by the substrate stage, and / or the chamber may be controlled.

[0037] In some examples, the at least two heating and / or cooling elements enable a thermal gradient to be established between different portions of the lower and / or the upper plate. In some examples, the established thermal gradient enables a specimen disposed on a substrate and / or a portion of the substrate to be maintained at a temperature less than the temperature of any other component within the chamber during an unmasking operation. For example, it is possible to independently operate the various heating and / or cooling elements present in the lower and / or upper plates such that at least a portion of the substrate, or the specimen disposed on the substrate, remains the coldest component within the chamber during an unmasking operation. For example, the substrate or the specimen disposed on the substrate may be maintained at a temperature lower than the upper plate, the lower plate, ports, valves, and / or any other structure within a chamber formed from the upper and lower plates during an unmasking operation.

[0038] In some examples, a duration of an unmasking operation is standardized for all types of specimens and all types of analyses to be performed on a given type of specimen, thereby increasing the efficiency of the system and all components therein. For example, having a standardized duration for all specimen and analysis types permits easier scheduling of prior specimen processing steps (such as deparaffinization) and subsequent specimen processing steps (such as staining steps) since they can all be in "lock step" with the standardized unmasking operation duration. Standardization of the unmasking operation duration is made possible by the discovery that one need only change the temperature to vary the extent of unmasking for a given type of sample. By selecting a specific temperature at which the unmasking operation is performed for a particular sample type, not only can the duration of the unmasking operation be standardized, but also the extent of unmasking for the particular sample type can be optimized for a particular assay. In some examples, the disclosed system can be optimized to facilitate standardization of the duration of the unmasking operation. Thus, in some examples, an unmasking operation in each independently operable chamber is conducted for the same duration of time regardless of the biomarkers being unmasked in any individual chamber.

[0039] Another aspect of the present disclosure is a system including one or more independently operable specimen processing assemblies, wherein the one or more independently operable specimen processing assemblies are adapted to independently treat and / or move a specimen bearing substrate from one processing area to another while maintaining the specimen bearing substrate in a horizontal position during all steps of processing (e.g. during treatment with one or more fluids and / or reagents; and during an unmasking operation). In some examples, the horizontal processing (and / or horizontal movements) enables the one or more fluids and / or reagents dispensed on the specimen bearing substrate to be maintained and / or manipulated on the surface of the substrate. In some examples, the one or more specimen processing assemblies include a chamber configured to process the specimen bearing substrates at an elevated temperature and / or pressure (as compared with temperatures and / or pressures external to the chamber), while maintaining the substrate in a horizontal position and while minimizing evaporative losses. In some examples, the chamber is formed from a lower plate having a lower engagement surface and an upper plate having an upper engagement surface, where the upper engagement surface is complementary to the lower engagement surface.

[0040] In some examples, the chamber is formed from a modular lower plate. In some examples, a modular lower plate includes a body having a lower engagement surface. In some examples, the modular lower plate includes a thermal management module. In some examples, the body may rest on, engage with, or couple to the thermal management module. In some examples, the body includes a substrate stage that is part of and integral with body. In other examples, the body includes a lower engagement surface but does not include an integral substrate stage. Rather, the modular lower plate includes a body having a separable substrate stage. In this way, a separable substrate stage may be used as a carrier for a substrate. For example, a substrate may be disposed onto the separable substrate stage and together the substrate and the separable substrate stage pair may be picked up, transported to, and deposited onto a body. In some examples, a substrate and a separate substrate stage pair remain together throughout all or at least some of the processing steps, for example, through all or part of the steps used to prepare a sample for microscopic analysis, such as from baking through coverslipping or from baking through staining. In some examples, a user places a substrate bearing a sample onto the separate substrate stage and inputs the substrate / substrate stage pair into a system and following processing of the sample, retrieves the substrate / substrate stage from the system, and then removes the substrate with a processed sample thereon for analysis.

[0041] In some examples, the lower and upper plates are independently moved together such that a sealing engagement is made between the complementary lower and upper engagement surfaces. In some examples, the formed chamber is configured for performing one or more steps of an unmasking operation, such any of the steps of heating, pressurizing, cooling, depressurizing, quenching, and / or adding or removing fluids and / or reagents /

[0042] In some examples, one or more fluids and / or reagents are dispensed to a specimen disposed on a substrate, a portion of the substrate itself, and / or a reservoir within the lower plate prior to the formation of the sealed chamber. By way of example, a total volume of between about 250µL to about 1000µL of one or more fluids and / or reagents are dispensed to a specimen disposed on a substrate, a portion of the substrate itself, and / or a reservoir within the lower plate before the formation of the sealed chamber. In some examples, an unmasking operation is performed using fluids and / or reagents supplied to the substrate prior to the formation of the chamber. For example, no additional fluids and / or reagents are dispensed to the substrate while the substrate is positioned within the chamber and during an unmasking operation.

[0043] In some examples, the chamber is communication with one or more independently operable heating and / or cooling elements which enable a substrate, a specimen, 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 examples, the heating of the fluid (such as a fluid disposed on the substrate or within a separate reservoir within the chamber) enables the chamber to be heated and / or pressurized. For example, the heating of the fluid may enable an unmasking operation to be conducted within the sealed chamber and on a specimen disposed on a substrate. In some examples, heating and / or pressurization of the chamber is monitored with 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 examples, heating is achieved with a conductive heating element positioned on or in the substrate stage, a conductive heating element in the body of one or both of the upper and lower plates, other types of heating devices in locations adjacent to the fluids and / or reagents being heated, using microwaves passed into the reaction compartment to heat the regents, and / or magnetic induction. In some examples, at least a portion of the specimen disposed on the substrate and / or at least a portion of the substrate itself is maintained at a temperature which is lower than any other component within the chamber during an unmasking operation. For example, a specimen disposed on the substrate or the substrate itself may be maintained as the "coldest" component within the chamber during an unmasking operation.

[0044] In some examples, 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 between about 115°C to about 155°C and at a pressure ranging from between about 150 kPa to about 1050 kPa for a time period ranging from between about 2 minutes to about 10 minutes. In some examples, the specimen and / or a portion of the substrate are maintained as the "coldest" components within the chamber throughout the entire heating and / or pressurization process.

[0045] In some examples, the unmasking operation comprises a temperature ramp-up phase, a temperature maintenance phase, and a temperature ramp-down phase. In some examples, a substrate disposed on a substrate or a portion of the substrate itself is maintained as the "coldest" component within the chamber during the temperature ramp-up phase, the temperature maintenance phase, and the temperature ramp-down phase. In some examples, the unmasking operation does not include a temperature ramp-down phase. For example, the sealed chamber may be opened while heated and / or pressurized. In some examples, some of the pressure is relieved through one or more valves in communication with the chamber prior to opening the heated and / or pressurized chamber.

[0046] In some examples, additional fluids and / or reagents are added to the specimen and / or the substrate during one or more of the temperature ramp-up phase, the temperature maintenance phase, and the temperature ramp-down phase. By way of example, between about 250µL to about 1000µL of one or more additional fluids and / or reagents are dispensed to the specimen or a portion of the substrate itself after the chamber is formed. In some examples, the one or more additional fluids and / or reagents dispensed into the sealed chamber have a temperature less than the temperature inside the heated and / or pressurized chamber. For example, the one or more additional fluids and / or reagents may have a temperature of at least 25°C less than the temperature of the specimen or the temperature inside the sealed chamber. In some examples, the entire sealed chamber is substantially filled with one or more liquids. For example, if the total chamber volume is about 2mL (not accounting for the volume of the substrate with the chamber), then about 2mL of additional fluids and / or reagents may be added while the chamber is sealed. In some examples, between about 250µL to about 1000µL of one or more additional fluids and / or reagents are dispensed to a reservoir within the chamber, such as a fluid reservoir which is heated. In alternate examples, the fluid reservoir is maintained as the coldest component with the chamber.

[0047] In some examples, the chamber is in communication with one or more ports which facilitate the delivery of one or more gases and / or steam into the chamber so as to pressurize the chamber and / or to heat a substrate, a specimen, and / or one or more fluids and / or reagents disposed within the chamber. In some examples, the lower plate may include one or more ports, e.g. vacuum ports, for removing excess liquids from within the chamber. In some examples, the vacuum ports in the lower plate may be activated after substantially filling the chamber with additional fluids and / or reagents.

[0048] Another aspect of the present disclosure is a system including (a) one or more independently operable specimen processing assemblies, wherein the one or more independently operable specimen processing assemblies includes (i) a lower plate, wherein the lower plate includes a lower engagement surface and one or more substrate stages raised relative to the lower engagement surface, and wherein the one or more raised substrate stages includes an upper surface adapted to hold a substrate horizontally; and (ii) an upper plate having an upper engagement surface complementary to the lower engagement surface, and wherein the upper plate further includes 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 raised substrate stage; (b) one or more dispense devices; and (c) a control system communicatively coupled to at least the one or more independently operable specimen processing assemblies and / or the one or more dispense devices.

[0049] In some examples, 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 examples, at least one heating and / or cooling element is embedded within a body of the lower plate; and at least one heating and / or cooling element is embedded within a body of the upper plate. In some examples, the control system is in communication with the one or more independently operable heating and / or cooling elements. In some examples, any of the heating and / or cooling elements within the upper plate may be operated in conjunction with those heating and / or cooling elements 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 a substrate supported by the substrate stage, and / or the chamber may be controlled. For example, it is possible to independently operate the various heating and / or cooling elements present in the lower and / or upper plates such that at least a portion of the substrate, or the specimen disposed on the substrate, remains the coldest structure within the chamber. For example, the substrate or the specimen disposed on the substrate may be maintained at a temperature lower than the upper plate, the lower plate, ports, valves, and / or any other structure within the chamber formed from the upper and lower plates. In some examples, the heating and / or cooling elements are each independently operated so as to maintain a predetermined temperature gradient between different portions of the upper and lower plates and the substrate stage.

[0050] In some examples, the one or more independently operable heating and / or cooling elements are in thermal communication with the one or more substrate stages. In some examples, the independently operable heating and / or cooling elements are configured such that a specimen disposed on a substrate and positioned on the surface of the substrate stage has a temperature which is lower than any other component in thermal communication with the one or more independently operable heating and / or cooling elements.

[0051] In some examples, each dispense device of the one or more dispense devices includes one or more dispense nozzles, one or more pipettes, and / or or one or more dispense-on-demand devices. For example, the dispense devices may be commanded by the control system to dispense a total volume of one or more fluids and / or reagents ranging from between about 250µL to about 1000µL. In some examples, the system further includes one or more mixing devices and / or one or more liquid removal devices. In some examples, the one or more dispense devices are coupled to a dispense sub-assembly. In some examples, the system includes a plurality of specimen processing assemblies. In some examples, at least one of the plurality of specimen processing assemblies includes a chamber formed from an upper plate and a lower plate. In some examples, a substrate is horizontally disposed within the chamber.

[0052] Another aspect of the present disclosure is a system including: (a) a plurality of independently operable specimen processing assemblies, wherein each independently operable specimen processing assembly includes (i) a lower plate movably coupled to a lower rail, wherein the lower plate includes a lower engagement surface and one or more substrate stages raised relative to the lower engagement surface, and wherein the one or more raised substrate stages includes an upper surface adapted to hold a substrate horizontally as the lower plate traverses the lower rail; and (ii) an upper plate having an upper engagement surface complementary to the lower engagement surface, and wherein the upper plate further includes 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 raised substrate stages; and wherein the lower plate is configured such that the planar upper surface of the substrate stage remains horizontal while the lower plate traverses the lower rail; (b) one or more dispense devices; and (c) a control system in communication with the plurality of independently operable specimen processing assemblies and / or the one or more dispense devices.

[0053] In some examples, the control system is adapted to maintain an environment within a chamber formed from the complementary lower and upper plates, such as by controlling one or more heating elements, one or more cooling elements, and / or one or more gas and / or steam generation and delivery units in communication with the chamber. In some examples, the one or more heating and / or cooling elements are independently controlled such that the specimen disposed on the substrate and / or a portion of the substrate itself is maintained at a temperature which is less than any other component (e.g. ports, valves, sensors, probes, etc.) within the formed chamber. In some examples, 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 such that a predetermined temperature and / or a predetermined pressure is maintained. For example, the temperature within the chamber may be monitored with a temperature sensor communicatively coupled to the control system. When the temperature within the chamber meets a predetermined threshold chamber temperature, the control system may command the one or more heating elements to either maintain a steady-state or turn off.

[0054] In some examples, the control system commands one or more force generating members to exert a predetermined external force onto at least one of an upper plate or a lower plate so as to maintain the upper and lower engagement surfaces of the upper and lower plates, respectively, in sealing engagement, especially when the pressure within any formed chamber is increased. In some examples, the control system commands any force generating member to apply a predetermined amount of force which is less than a force generated at a predetermined threshold pressure. In some examples, the control system may command one or more ports and / or valves to release pressure above a predetermined threshold pressure.

[0055] In some examples, the one or more substrate stages are configured such that any substrate supported by the one or more substrate stages are held in a horizontal position and remains in the horizontal position during the 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 a completely vertical movement).

[0056] In some examples, at least one of the specimen processing assemblies includes a sealed chamber. In some examples, the system further includes a substrate at least partially disposed within the sealed chamber, and wherein the substrate is supported by the one or more substrate stages and is oriented in a horizontal position. In some examples, a specimen disposed on the substrate is treated outside the chamber with one or more fluids and / or reagents while in a horizontal position and wherein an unmasking operation is performed within the chamber while the substrate is disposed in a horizontal position. In some examples, no additional fluids and / or reagents are dispensed to the substrate while the substrate is positioned within the chamber. In some examples, an unmasking operation is conducted using only the fluids and / or reagents dispensed to the substrate prior to the formation of the chamber. In some examples, a specimen disposed on the substrate or the substrate itself is maintained as the "coldest" component within the chamber during all phases on an unmasking operation. For example, the substrate and / or a portion of the substrate are maintained as the "coldest" component within the chamber during a temperature ramp-up phase, a temperature maintenance phase, and a temperature ramp-down phase of an unmasking operation.

[0057] In some examples, the lower rail of each of the plurality of independently operable specimen processing assemblies is positioned horizontally, for example each lower rail is arranged parallel to the ground. In some examples, the lower rail of each of the plurality of independently operable specimen processing assemblies includes a first end which is raised vertically relative to a second end, such that each lower rail deviates from horizontal, for example the rail is offset from the horizontal.

[0058] In some examples, the system further includes a motor adapted to move the lower plate from a first position along the lower rail to a second position along the lower rail. In some examples, the second position along the lower rail is beneath the upper plate, e.g. a predetermined position beneath a pre-positioned upper plate. In some examples, the second position along the first lower rail is in a preparation area and wherein the upper plate is moved (along any of the x, y, and z directions) to the pre-positioned lower plate.

[0059] In some examples, the dispense device is coupled to a dispense rail, wherein the dispense rail oriented perpendicular to the lower rail of each of the plurality of independently operable specimen processing assemblies (regardless of whether the lower rails are arranged horizontally or offset from the horizontal). In some examples, the upper plate is coupled to a force generating member. In some examples, 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 examples, the upper plate is fixed to a sub-assembly.

[0060] In some examples, the disclosed system includes at least two independently operable specimen processing assemblies. In some examples, one of the at least two independently operable specimen processing assemblies includes a chamber.

[0061] Another aspect of the present disclosure is a specimen processing assembling including (i) a lower plate including (a) a substrate stage having an upper surface adapted to support a substrate horizontally, (b) a lower engagement surface, wherein the lower engagement surface at least partially circumscribes the substrate stage, and (c) a first lower temperature regulation element in thermal communication with the substrate stage; and (ii) an upper plate including (a) an upper engagement surface complementary to the lower engagement surface, and (b) a cavity. In some examples, the first lower temperature regulation element is positioned beneath the substrate stage. In some examples, the lower plate further includes a second lower temperature regulation element and a third lower temperature regulation element. In some examples, each of the temperature regulation elements are independently operable. In some examples, the second and third lower temperature regulation elements are each positioned adjacent the first lower temperature regulation element. In some examples, the second and third lower temperature regulation elements are each positioned beneath a portion of the lower engagement surface. In some examples, the first, second, and third lower engagement elements are positioned in parallel to each other.

[0062] In some examples, a thermal gradient is maintained between the first and second lower temperature regulation elements and between the first and third temperature regulation elements. In some examples, the thermal gradient maintained between the first and second lower temperature regulation elements ranges from between 2°C to about 10°C; and wherein the thermal gradient maintained between the first and second lower temperature regulation elements ranges from between 2°C to about 10°C. In some examples, the thermal gradient maintained between the first and second lower temperature regulation elements ranges from between 2°C to about 5°C; and wherein the thermal gradient maintained between the first and second lower temperature regulation elements ranges from between 2°C to about 5°C. In some examples, wherein the first, second, and third lower temperature regulation elements are positioned to maintain a thermal gradient at least between portions of the lower engagement surface and the substrate stage.

[0063] In some examples, the first lower temperature regulation element has a first thermal output, the second lower temperature regulation element has a second thermal output, and the third lower temperature regulation element has a third thermal output, wherein the first thermal output is less than either of the second and third thermal outputs. In some examples, the upper plate further includes at least one upper thermal regulation element. In some examples, a thermal output of the first lower temperature regulation element is maintained at a temperature less than a thermal output of the at least one upper thermal regulation element.

[0064] In some examples, the specimen processing assembly further includes a substrate disposed on the surface of substrate stage. In some examples, the substrate is maintained at a temperature which is less than the temperature of the lower and upper engagement surfaces. In some examples, a specimen disposed on the substrate or a portion of the substrate is maintained at a temperature which is less than any other component within the chamber, for example the specimen disposed on the substrate or the portion of the substrate is the "coldest" component within the chamber during an unmasking operation.

[0065] In some examples, the first lower temperature regulation element includes at least one fluid channel. In some examples, the second and third lower temperature regulation elements each comprise heating cartridges. In some examples, at least one of the lower plate and the upper plate includes at least one seal body. In some examples, the at least one seal body is removable. In some examples, 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.

[0066] In some examples, the specimen processing assembly further includes a sub-assembly having first and second ends. In some examples, the lower plate is movable along a length of the sub-assembly between the first and second ends.

[0067] In some examples, the sub-assembly is arranged horizontally. In some examples, the lower plate and upper plate are both moved independently to predetermined positions such that the lower engagement surface of the lower plate at least partially contacts the complementary upper engagement surface of the upper plate. In some examples, the lower plate is moved to a predetermined position and then the upper plate is moved toward the lower plate. In some examples, the movement toward the lower plate is performed using one of a motor, a piston, or a cam mechanism.

[0068] In some examples, the sub-assembly is offset from the horizontal. In some examples, the upper plate is held stationary and wherein the lower plate is moved toward the upper plate until the lower engagement surface of the lower plate at least partially contacts the complementary upper engagement surface of the upper plate. In some examples, the upper plate and lower plate have complementary wedge-based shapes. In some examples, the lower plate and upper plate are both moved independently to predetermined positions such that the lower engagement surface of the lower plate at least partially contacts the complementary upper engagement surface of the upper plate. In some examples, the lower and upper plates are both independently movable.

[0069] Another aspect of the present disclosure is a method of unmasking a specimen disposed on a substrate including: (a) dispensing a predetermined volume of one or more fluids and / or reagents to at least a portion of the specimen; (b) sealing the specimen within a chamber, wherein the chamber is formed by contacting (i) a lower engagement surface of a lower plate, with (ii) 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 includes a substrate stage for supporting the substrate, and (c) performing an unmasking operation on the specimen within the sealed chamber. In some examples, the lower plate includes one or more independently operable heating and / or cooling elements. In some examples, the upper plate includes one or more independently operable heating and / or cooling elements. In some examples, both the lower and upper plates include operable heating and / or cooling elements. In some examples, any of the heating and / or cooling elements within the upper plate may be operated in conjunction with those heating and / or cooling elements 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 a substrate supported by the substrate stage, and / or the chamber may be controlled.

[0070] In some examples, the chamber is formed from a lower plate having a modular design. In some examples, a modular lower plate includes a body having a lower engagement surface. In some examples, the modular lower plate includes a thermal management module. In some examples, the body may rest on, engage with, or couple to the thermal management module. In some examples, the body includes a substrate stage that is part of and integral with body. In other examples, the body includes a lower engagement surface but does not include an integral substrate stage. Rather, the modular lower plate includes a body having a separable substrate stage. In this way, a separable substrate stage may be used as a carrier for a substrate. For example, a substrate may be disposed onto the separable substrate stage and together the substrate and the separable substrate stage pair may be picked up, transported to, and deposited onto a body. In some examples, a substrate and a separate substrate stage pair remain together throughout all or at least some of the processing steps, for example, through all or part of the steps used to prepare a sample for microscopic analysis, such as from baking through coverslipping or from baking through staining. In some examples, a user places a substrate bearing a sample onto the separate substrate stage and inputs the substrate / substrate stage pair into a system and following processing of the sample, retrieves the substrate / substrate stage from the system, and then removes the substrate with a processed sample thereon for analysis.

[0071] In some examples, the one or more fluids and / or reagents are selected from water and a buffer solution having a pH ranging from between about 5 to about 10. In some examples, the one or more fluids and / or reagents comprise a mixture of deionized water, tris(hydroxymethyl)methylamine, and a chelator. In some examples, the predetermined volume of the one or more fluids and / or reagents dispensed to the at least a portion of the specimen ranges from between about 200µL to about 1000µL. In some examples, the predetermined volume of the one or more fluids and / or reagents dispensed to the at least the portion on\f the specimen ranges from between about 250µL to about 500µL.

[0072] In some examples, the unmasking operation includes the step of heating the specimen disposed on the substrate to a first predetermined temperature for a predetermined duration of time. In some examples, the first predetermined temperature ranges from between about 125°C to about 155°C. In some examples, the first predetermined temperature ranges from between about 135°C to about 150°C. In some examples, the first predetermined temperature is about 140°C. In some examples, the predetermined duration of time ranges from between about 1 minute to about 10 minutes. In some examples, the predetermined duration of time ranges from between about 1 minute to about 7 minutes. In some examples, the predetermined duration of time ranges from between about 1 minute to about 5 minutes.

[0073] In some examples, a temperature of the substrate is less than a temperature of any other component within the chamber during the unmasking operation. In some examples, the temperature of the substrate is at least 10°C less than the temperature of the other components within the chamber. In some examples, the temperature of the substrate is at least 5°C less than the temperature of the other components within the chamber.

[0074] In some examples, less than about 5% of the predetermined volume of the one or more fluids and / or reagents dispensed to the specimen is lost to evaporation during the unmasking operation. In some examples, less than about 2% of the predetermined volume of the one or more fluids and / or reagents dispensed to the specimen is lost to evaporation during the unmasking operation. In some examples, substantially no further fluids and / or reagents are dispensed to the substrate after the chamber is sealed. In some examples, no further fluids and / or reagents are dispensed to the substrate after the chamber is sealed.

[0075] In some examples, the method further includes pre-pressurizing the sealed chamber. In some examples, the method further includes introducing steam into the sealed chamber.

[0076] In some examples, the lower plate further includes at least one heating element. In some examples, the lower plate further includes at least three heating elements, wherein a first of the at least three heating elements is positioned beneath the substrate stage, and wherein the second and third of the at least three heating elements are each positioned adjacent the first of the at least three heating elements. In some examples, a first thermal gradient is maintained between the first and second of the at least three heating elements during the performance of the unmasking operation, and wherein a second thermal gradient is maintained between the second and third of the at least three heating elements during the performance of the unmasking operation.

[0077] In some examples, a force generating member applies an external force to the sealed chamber. In some examples, the force generating member is selected from the group consisting a motor, a piston, a spring, a screw mechanism, a lever, a cam mechanism. In some examples, at least one of the lower and upper plates is in thermal communication with a thermal management module. In some examples, the unmasking operation includes a temperature ramp-up phase and a temperature maintenance phase. In some examples, the temperature ramp-up phase includes heating the specimen at a rate ranging from between about 1°C / s to about 4°C / s. In some examples, the temperature ramp-up phase includes heating the specimen at a rate ranging from between about 3°C / s to about 4°C / s.

[0078] In some examples, unmasking operation further includes a temperature ramp-down phase. In some examples, the temperature ramp-up phase is shorter than the temperature ramp-down phase. In some examples, the temperature ramp-down phase includes cooling the specimen at a rate ranging from between about 0.5°C / s to about 3°C / s.

[0079] In some examples, the unmasking operation is stopped by opening the chamber without a temperature ramp-down phase. In some examples, the unmasking operation is stopped by introducing a predetermined amount of a fluid into the chamber. In some examples, the predetermined amount of fluid introduced to stop the unmasking operation ranges from between about 0.5mL to about 5mL.

[0080] In some examples, the method further includes staining the specimen for the presence of one or more biomarkers after the completion of the unmasking operation.

[0081] Another aspect of the present disclosure is a method of unmasking a specimen disposed on a substrate including: (a) dispensing a predetermined volume of one or more fluids and / or reagents to at least a portion of the specimen; (b) sealing the specimen within a chamber, wherein the chamber includes (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 by the substrate stage in a horizontal position; and (c) performing an unmasking operation within the sealed chamber. In some examples, the one or more fluids and / or reagents dispensed to the specimen are selected from the group consisting of water and a buffer solution. In some examples, the predetermined volume of the one or more fluids and / or reagents dispensed to the specimen ranges from between about 200µL to about 1000µL. In some examples, the predetermined volume of the one or more fluids and / or reagents dispensed to the specimen ranges from between about 250µL to about 500µL.

[0082] In some examples, the lower plate includes one or more independently operable heating and / or cooling elements. In some examples, the upper plate includes one or more independently operable heating and / or cooling elements. In some examples, both the lower and upper plates include independently operable heating and / or cooling element. In some examples, any of the heating and / or cooling elements within the upper plate may be operated in conjunction with those heating and / or cooling elements 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 a substrate supported by the substrate stage, and / or the chamber may be controlled.

[0083] In some examples, the lower plate has a modular design. In some examples, a modular lower plate includes a body having a lower engagement surface. In some examples, the modular lower plate includes a thermal management module. In some examples, the body may rest on, engage with, or couple to the thermal management module. In some examples, the body includes a substrate stage that is part of and integral with body. In other examples, the body includes a lower engagement surface but does not include an integral substrate stage. Rather, the modular lower plate includes a body having a separable substrate stage. In this way, a separable substrate stage may be used as a carrier for a substrate. For example, a substrate may be disposed onto the separable substrate stage and together the substrate and the separable substrate stage pair may be picked up, transported to, and deposited onto a body. In some examples, a substrate and a separate substrate stage pair remain together throughout all or at least some of the processing steps, for example, through all or part of the steps used to prepare a sample for microscopic analysis, such as from baking through coverslipping or from baking through staining. In some examples, a user places a substrate bearing a sample onto the separate substrate stage and inputs the substrate / substrate stage pair into a system and following processing of the sample, retrieves the substrate / substrate stage from the system, and then removes the substrate with a processed sample thereon for analysis.

[0084] In some examples, the unmasking operation includes heating the specimen disposed on the substrate to a first predetermined temperature for a predetermined duration of time. In some examples, the first predetermined temperature ranges from between about 125°C to about 155°C. In some examples, the predetermined duration of time ranges from between about 1 minute to about 10 minutes. In some examples, a temperature of the substrate is less than a temperature of any other component within the chamber during the unmasking operation. In some examples, the temperature of the substrate is at least 5°C less than the temperature of the other components within the chamber. In some examples, less than about 5% of the predetermined volume of the one or more fluids and / or reagents dispensed to the specimen are lost to evaporation during the unmasking operation. In some examples, no further fluids and / or reagents are dispensed to the substrate after the chamber is sealed.

[0085] In some examples, the method further includes pre-pressurizing the sealed chamber prior to the performance of the unmasking operation. In some examples, the chamber is pre-pressurized simultaneously with the performance of the unmasking operation. In some examples, the method further includes introducing steam into the sealed chamber.

[0086] In some examples, the unmasking operation includes heating the specimen at a rate ranging from between about 1°C / s to about 4°C / s. In some examples, the unmasking operation includes heating the specimen at a rate ranging from between about 3°C / s to about 4°C / s. In some examples, the unmasking operation includes cooling the specimen at a rate ranging from between about 0.5°C / s to about 2.5°C / s.

[0087] In another aspect of the present disclosure is a method of unmasking a specimen disposed on a substrate including: (a) dispensing a predetermined volume of one or more fluids and / or reagents to at least a portion of the specimen; (b) sealing the specimen 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 by the substrate stage in a horizontal position; and (c) performing an unmasking operation, wherein the unmasking operation includes a temperature ramp-up phase, a temperature maintenance phase, and a temperature ramp-down phase. In some examples, the temperature ramp-up phase includes heating the specimen at a rate ranging from between about 1°C / s to about 4°C / s. In some examples, temperature ramp-up phase includes heating the specimen at a rate ranging from between about 3°C / s to about 4°C / s. In some examples, the temperature ramp-up phase includes heating the specimen to a predetermined temperature of between about 110°C to about 150°C. In some examples, the temperature ramp-up phase includes heating the specimen to a predetermined temperature of between about 120°C to about 145°C.

[0088] In some examples, the specimen is maintained at the predetermined temperature for a predetermined time period ranging from between about 1 minute to about 10 minutes. In some examples, specimen is maintained at the predetermined temperature for a predetermined time period ranging from between about 2 minutes to about 7 minutes. In some examples, the specimen is maintained at the predetermined temperature for a predetermined time period ranging from between about 3 minutes to about 5 minutes.

[0089] In some examples, the temperature ramp-down phase includes cooling the specimen at a rate ranging from between about 0.5°C / s to about 3°C / s. In some examples, temperature ramp-down phase includes cooling the specimen at a rate ranging from between about 0.5°C / s to about 2°C / s.

[0090] In some examples, the one or more fluids and / or reagents are selected from the group consisting of water and a buffer solution. In some examples, the predetermined volume of the one or more fluids and / or reagents dispensed to the specimen ranges from between about 200µL to about 1000µL. In some examples, the predetermined volume of the one or more fluids and / or reagents dispensed to the specimen ranges from between about 250µL to about 500µL.

[0091] Another aspect of the present disclosure is an antigen retrieved specimen prepared by (a) dispensing a predetermined volume of one or more fluids and / or reagents to at least a portion of the specimen; (b) sealing the specimen 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 by the substrate stage in a horizontal position; and (c) performing an unmasking operation within the sealed chamber.

[0092] Another aspect of the present disclosure is a target retrieved specimen prepared by (a) dispensing a predetermined volume of one or more fluids and / or reagents to at least a portion of the specimen; (b) sealing the specimen 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 by the substrate stage in a horizontal position; and (c) performing an unmasking operation within the sealed chamber.

[0093] Another aspect of the present disclosure is a target retrieved specimen prepared by (a) dispensing a predetermined volume of one or more fluids and / or reagents to a portion of the specimen; (b) sealing the specimen within a chamber, where 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 includes a substrate stage for supporting the substrate; and (c) performing an unmasking operation on the specimen within the sealed chamber. In some examples, the antigen retrieved specimen is a biopsy sample.

[0094] Another aspect of the present disclosure is an antigen retrieved specimen prepared by (a) dispensing a predetermined volume of one or more fluids and / or reagents to at least a portion of the specimen; (b) sealing the specimen 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 by the substrate stage in a horizontal position; and (c) performing an unmasking operation, wherein the unmasking operation includes a temperature ramp-up phase, a temperature maintenance phase, and a temperature ramp-down phase. In some examples, the specimen is retained as the "coldest" component within the sealed chamber during performance of all phases of the unmasking operation, for example during a temperature ramp-up phase, during a temperature maintenance phase, and during a temperature ramp-down phase.

[0095] Another aspect of the present disclosure is a target retrieved specimen prepared by (a) dispensing a predetermined volume of one or more fluids and / or reagents to at least a portion of the specimen; (b) sealing the specimen 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 by the substrate stage in a horizontal position; and (c) performing an unmasking operation, wherein the unmasking operation includes a temperature ramp-up phase, a temperature maintenance phase, and a temperature ramp-down phase. In some examples, the specimen is retained as the "coldest" component within the sealed chamber during performance of all phases of the unmasking operation, for example during a temperature ramp-up phase, during a temperature maintenance phase, and during a temperature ramp-down phase.

[0096] Another aspect of the present disclosure is an unmasked specimen disposed on a substrate, wherein the unmasked specimen is prepared according to a process including (a) dispensing a predetermined volume of one or more fluids and / or reagents to a portion of a specimen disposed on the substrate; (b) sealing the specimen within a chamber, where 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 includes a substrate stage for supporting the substrate; and (c) performing an unmasking operation on the specimen within the sealed chamber. In some examples, the antigen retrieved specimen is a biopsy sample, and (c) performing an unmasking operation on the specimen. In some examples, the unmasked specimen includes one or more retrieved antigenic sites. In some examples, the unmasked specimen includes one or more retrieved nucleic acid targets. In some examples, no further fluids and / or reagents are dispensed to the substrate after the chamber is sealed.

[0097] In some examples, the lower plate includes one or more independently operable heating and / or cooling elements. In some examples, the upper plate includes one or more independently operable heating and / or cooling elements. In some examples, both the lower and upper plates include independently operable heating and / or cooling element. In some examples, any of the heating and / or cooling elements within the upper plate may be operated in conjunction with those heating and / or cooling elements 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 a substrate supported by the substrate stage, and / or the chamber may be controlled.

[0098] In some examples, the lower plate has a modular design. In some examples, a modular lower plate includes a body having a lower engagement surface. In some examples, the modular lower plate includes a thermal management module. In some examples, the body may rest on, engage with, or couple to the thermal management module. In some examples, the body includes a substrate stage that is part of and integral with body. In other examples, the body includes a lower engagement surface but does not include an integral substrate stage. Rather, the modular lower plate includes a body having a separable substrate stage. In this way, a separable substrate stage may be used as a carrier for a substrate. For example, a substrate may be disposed onto the separable substrate stage and together the substrate and the separable substrate stage pair may be picked up, transported to, and deposited onto a body.

[0099] In some examples, a substrate and a separate substrate stage pair remain together throughout all or at least some of the processing steps, for example, through all or part of the steps used to prepare a sample for microscopic analysis, such as from baking through coverslipping or from baking through staining. In some examples, a user places a substrate bearing a sample onto the separate substrate stage and inputs the substrate / substrate stage pair into a system and following processing of the sample, retrieves the substrate / substrate stage from the system, and then removes the substrate with a processed sample thereon for analysis. In some examples, the one or more fluids and / or reagents are selected from the group consisting of water and a buffer solution having a pH ranging from between about 5 to about 10. In some examples, the one or more fluids and / or reagents comprise a mixture of deionized water, tris(hydroxymethyl)methylamine, and a chelator. In some examples, the predetermined volume of the one or more fluids and / or reagents dispensed to the specimen ranges from between about 200µL to about 1000µL. In some examples, the predetermined volume of the one or more fluids and / or reagents dispensed to the specimen ranges from between about 250µL to about 500µL.

[0100] In some examples, the unmasking operation includes heating the specimen disposed on the substrate to a first predetermined temperature for a predetermined duration of time. In some examples, the first predetermined temperature ranges from between about 125°C to about 155°C. In some examples, the first predetermined temperature ranges from between about 135°C to about 150°C. In some examples, the first predetermined temperature is about 140°C. In some examples, the predetermined duration of time ranges from between about 1 minute to about 10 minutes. In some examples, the predetermined duration of time ranges from between about 1 minute to about 5 minutes.

[0101] In some examples, a temperature of the substrate is less than a temperature of any other component within the chamber during the unmasking operation. In some examples, the temperature of the substrate is at least 5°C less than the temperature of the other components within the chamber. In some examples, less than about 5% of the predetermined volume of the one or more fluids and / or reagents dispensed to the specimen is lost to evaporation during the unmasking operation. In some examples, less than about 2% of the predetermined volume of the one or more fluids and / or reagents dispensed to the specimen is lost to evaporation during the unmasking operation.

[0102] In some examples, the sealed chamber is pre-pressurized. In some examples, steam is further introduced into the sealed chamber.

[0103] In some examples, the lower plate further includes at least one heating element. In some examples, the lower plate further includes at least three heating elements, wherein a first of the at least three heating elements is positioned beneath the substrate stage, and wherein the second and third of the at least three heating elements are positioned adjacent the first of the at least three heating elements. In some examples, a first thermal gradient is maintained between the first and second of the at least three heating elements during the performance of the unmasking operation, and wherein a second thermal gradient is maintained between the second and third of the at least three heating elements during the performance of the unmasking operation.

[0104] Another aspect of the present disclosure is an unmasked specimen, where the unmasked specimen is prepared in a process where a specimen disposed on a substrate is retained as the coldest component within a sealed chamber during an unmasking operation, wherein unmasked specimen is prepared by: (a) dispensing a predetermined volume of one or more fluids and / or reagents to a portion of the specimen disposed on the substrate; (b) positioning the specimen bearing substrate including the dispensed one or more fluids and / or reagents onto a substrate stage within a chamber formed from an upper plate and a lower plate, wherein the lower plate includes the substrate stage and a lower engagement surface, and wherein the upper plate includes an upper engagement surface complementary to the lower engagement surface; (c) sealing the chamber; and (d) performing an unmasking operation on the specimen. In some examples, the unmasked specimen includes one or more retrieved antigenic sites. In some examples, the unmasked specimen includes one or more retrieved nucleic acid targets. In some examples, no further fluids and / or reagents are dispensed to the substrate after the chamber is sealed.

[0105] Another aspect of the present disclosure is directed to a system including_(i) at least one chamber, wherein the at least one chamber comprises an upper plate and a lower plate, wherein the lower plate includes a lower engagement surface and one or more substrate stages adapted to hold a substrate horizontally within the at least one chamber, and wherein the upper plate includes an upper engagement surface which is 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 includes at least one of a heating element or a cooling element; and (ii) a staining module. In some examples, the at least one chamber is an unmasking chamber.

[0106] In some examples, the at least one unmasking chamber has a predetermined internal volume. In some examples, the predetermined internal volume ranges from between about 14cm 3< to about 25cm 3< . In some examples, the predetermined internal volume ranges from between about 16cm 3< to about 22cm 3< . In some examples, the predetermined internal volume ranges from between about 18cm 3< to about 20cm 3< . In some examples, the one or more substrate stages are raised relative to the lower engagement surface. In some examples, the upper plate includes at least one steam injection port. In some examples, the at least one steam injection port is in fluidic communication with a steam reservoir or a steam generating element. In some examples, the at least one chamber is configured to rapidly heat and / or pressurize the predetermined interior volume. In some examples, the lower and / or upper plates may be separated from each other after the steam heating and / or pressurization.

[0107] In some examples, at least one of the lower and / or upper plates further comprises one or more alignment members. In some examples, at least one of the lower and / or upper plates further comprises one or more temperature and / or pressure sensors. In some examples, at least one of the lower and / or upper plates further comprises one or more temperature sensors which contact the horizontally held substrate or a fluid disposed thereon. In some examples, the one or more temperature sensors directly contact the horizontally held substrate or a fluid disposed thereon.

[0108] In some examples, a body of the lower plate and a body of the upper plate both have complementary wedge-based shapes. In some examples, at least one of the lower plate and the upper plate includes at least one seal body. In some examples, the at least one seal body is removable. In some examples, the lower plate comprises a groove and wherein the at least one removable seal body at least partially engages the groove. In some examples, the at least one removable seal body is integrated within a removable seal attachment, wherein the removable seal attachment engages a portion of a periphery of the lower plate or the upper plate.

[0109] In some examples, 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 examples, the upper and lower plates are independently movable.

[0110] In some examples, the system further includes a control system. In some examples, the control system is adapted to operate the at least one heating or cooling element to uniformly heat and / or cool the one or more substrate stages. In some examples, the control system is adapted to operate the at least one heating or cooling element to maintain the one or more substrate stages as the coolest component within the chamber.

[0111] In some examples, the system includes at least one substrate transfer device. In some examples, the at least one substrate transfer device is selected from the group consisting of a gripper device, a forklift device, and carrier transport. In some examples, the system further includes one or more substrate loading stations.

[0112] In some examples, the lower plate is modular. In some examples, the modular lower plate is transportable to the upper plate. In some examples, the upper plate is coupled to a force generating member. In some examples, the force generating member is configured to give way if a pressure within the chamber exceeds a predetermined threshold.

[0113] Another aspect of the present disclosure is a system including (i) at least one unmasking chamber having a predetermined interior volume, wherein the at least one unmasking chamber comprises an upper plate and a lower plate, wherein the lower plate includes a lower engagement surface and one or more substrate stages adapted to hold a substrate horizontally within the at least one unmasking chamber, and wherein the upper plate includes an upper engagement surface which is 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 within the at least one unmasking chamber; (ii) a staining module; and (iii) a steam reservoir. In some embodiments, a predetermined internal volume of the at least one unmasking chamber ranges from between about 14cm 3< to about 25cm 3< . In some embodiments, the upper and lower plates may be removably coupled together (e.g. clamped together) such that upon reaching a predetermining internal pressure and / or temperature, the coupling may be disengaged, and the upper and lower plates may rapidly separate from each other. In some embodiments, at least one of the lower and / or upper plate 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 may be operated in conjunction with the one or more steam injection ports.

[0114] In some embodiments, the upper plate is coupled to a force generating member. In some embodiments, the force generating member is configured to give way if a 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.

[0115] In some embodiments, at least one of the lower and / or upper plates further comprises one or more alignment members.

[0116] In some embodiments, at least one of the lower and / or upper plates further comprise one or more temperature and / or pressure sensors. At least one of the lower plate and the upper plate further comprises at least one seal body. In some embodiments, the at least one seal body is removable. In some embodiments, the lower plate comprises a groove and wherein the at least one removable seal body at least partially engages the groove.

[0117] Another aspect of the present disclosure is a system comprising: (i) at least one specimen processing assembly comprising: (1) a lower plate comprising a body, the body comprising (a) a substrate stage having an upper surface adapted to support a substrate horizontally, (b) a lower engagement surface, and (c) a first lower temperature regulation element in thermal communication with the substrate stage; and (2) an upper plate comprising an upper engagement surface complementary to the lower engagement surface; and (ii) a staining module.

[0118] In some examples, the first lower temperature regulation element is positioned beneath the substrate stage. In some examples, the lower plate further comprises a second lower temperature regulation element and a third lower temperature regulation element, wherein the second and third lower temperature regulation elements are each positioned adjacent the first lower temperature regulation element. In some examples, the first lower temperature regulation element has a first thermal output, the second lower temperature regulation element has a second thermal output, and the third lower temperature regulation element has a third thermal output, wherein the first thermal output is less than either of the second and third thermal outputs.

[0119] In some examples, a thermal gradient is maintained between the first and second lower temperature regulation elements and between the first and third temperature regulation elements. In some examples, the thermal gradient maintained between the first and second lower temperature regulation elements ranges from between 2°C to about 10°C; and wherein the thermal gradient maintained between the first and second lower temperature regulation elements ranges from between 2°C to about 10°C.

[0120] In some examples, the lower plate is modular. In some examples, the modular lower plate is transportable to an upper plate. In some examples, the upper plate includes an upper engagement surface which is complementary to the lower engagement surface and a cavity recessed relative to the upper engagement surface. In some examples, the upper plate is coupled to a force generating member. In some examples, the force generating member is configured to give way if a pressure within the chamber exceeds a predetermined threshold.

[0121] In some examples, 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.

[0122] In some examples, the system further comprises at least one substrate transfer device. In some examples, the at least one substrate transfer device is selected from the group consisting of a gripper device, a forklift device, and carrier transport. In some examples, the system further comprises one or more substrate loading stations.BRIEF DESCRIPTION OF THE FIGURES

[0123] For a general understanding of the features of the disclosure, reference is made to the drawings. In the drawings, like reference numerals have been used throughout to identify identical elements. FIG. 1A illustrates a top down view of a lower plate in accordance with one embodiment of the present disclosure. FIG. 1B illustrates a side view of a lower plate in accordance with one embodiment of the present disclosure. FIG. 1C illustrates a side view (longitudinal side) of a lower plate in accordance with one embodiment of the present disclosure. FIG. 1D illustrates a top down view of a lower plate including a vacuum port in accordance with one embodiment of the present disclosure. FIG. 1E illustrates a top down view of a lower plate including a vacuum port in accordance with one embodiment of the present disclosure. FIG. 1F illustrates a perspective view of a lower plate including a vacuum port in accordance with one embodiment of the present disclosure. FIG. 1G illustrates a top down view of a lower plate including a vacuum port in accordance with one embodiment of the present disclosure. FIG. 1H illustrates a top down view of a lower plate including a vacuum port in accordance with one embodiment of the present disclosure. FIG. 2A illustrates a top down view of a lower plate in accordance with one embodiment of the present disclosure. FIG. 2B illustrates a side view of a first end of a lower plate in accordance with one embodiment of the present disclosure. FIG. 2C illustrates a side view of a second end lower plate in accordance with one embodiment of the present disclosure. FIG. 2D illustrates a side view (longitudinal side) of a lower plate in accordance with one embodiment of the present disclosure. FIG. 3A illustrates a top down view of a lower plate in accordance with one embodiment of the present disclosure. FIG. 3B illustrates a side view (longitudinal side) of a lower plate in accordance with one embodiment of the present disclosure. FIG. 4A illustrates a top down view of a lower plate in accordance with one embodiment of the present disclosure. FIG. 4B illustrates a side view of a first end of a lower plate in accordance with one embodiment of the present disclosure. FIG. 4C illustrates a side view of a second end lower plate in accordance with one embodiment of the present disclosure. FIG. 4D illustrates a side view (longitudinal side) of a lower plate in accordance with one embodiment of the present disclosure. FIG. 5A illustrates a seal attachment removably coupled to a lower plate in accordance with one embodiment of the present disclosure. FIG. 5B illustrates a seal attachment positioned above a lower plate in accordance with one embodiment of the present disclosure. FIG. 6A illustrates a lower plate having a wedge-based shape in accordance with one embodiment of the present disclosure. FIG. 6B illustrates a lower plate having a wedge-based shape and illustrates an upper plate having a wedge-based shaped, wherein the upper and lower plates are in contact with one another in accordance with one embodiment of the present disclosure. FIG. 6C illustrates a side view of a first end of a lower plate having a wedge-based shaped in accordance with one embodiment of the present disclosure. FIG. 6D illustrates a side view of a second end of a lower plate having a wedge-based shaped in accordance with one embodiment of the present disclosure. FIG. 6E illustrates a side view of a lower plate having a wedge-based shaped in accordance with one embodiment of the present disclosure. FIG. 6F illustrates a side view of a lower plate having a wedge-based shaped in accordance with one embodiment of the present disclosure. FIG. 7A illustrates a lower plate coupled to a housing member in accordance with one embodiment of the present disclosure. FIG. 7B illustrates a lower plate coupled to a housing member in accordance with one embodiment of the present disclosure. FIG. 7C illustrates a lower plate coupled to a housing member in accordance with one embodiment of the present disclosure. FIG. 7D illustrates a lower plate coupled to a housing member in accordance with one embodiment of the present disclosure. FIG. 8A illustrates a lower plate coupled to a heat sink in accordance with one embodiment of the present disclosure. FIG. 8B illustrates a lower plate coupled to a heat sink in accordance with one embodiment of the present disclosure. FIG. 9A illustrates a top down view of an upper plate in accordance with one embodiment of the present disclosure. FIG. 9B illustrates a top down view of an upper plate in accordance with one embodiment of the present disclosure. FIG. 9C illustrates a side view of a first end of an upper plate in accordance with one embodiment of the present disclosure. FIG. 9D illustrates a side view of a first end of an upper plate in accordance with one embodiment of the present disclosure. FIG. 9E illustrates a side view of a first end of an upper plate in accordance with one embodiment of the present disclosure. FIG. 9F illustrates a side view of a first end of an upper plate in accordance with one embodiment of the present disclosure. FIG. 9G illustrates an upper plate including a heating element in accordance with one embodiment of the present disclosure. FIG. 9H illustrates an upper plate including a heating element in accordance with one embodiment of the present disclosure. FIG. 10A illustrates a lower plate and an upper plate in contact with each other in accordance with one embodiment of the present disclosure. FIG. 10B illustrates a lower plate and an upper plate in contact with each other in accordance with one embodiment of the present disclosure. FIG. 10C illustrates a lower plate and an upper plate in contact with each other in accordance with one embodiment of the present disclosure. FIG. 10D illustrates a lower plate and an upper plate in contact with each other in accordance with one embodiment of the present disclosure. FIG. 10E illustrates a lower plate and an upper plate in contact with each other in accordance with one embodiment of the present disclosure. FIG. 10F illustrates a lower plate and an upper plate in contact with each other in accordance with one embodiment of the present disclosure. FIG. 11A illustrates a lower plate having an integrated heat sink and an upper plate positioned above the lower plate in accordance with one embodiment of the present disclosure. FIG. 11B illustrates a lower plate having an integrated heat sink and an upper plate in contact with the lower plate in accordance with one embodiment of the present disclosure. FIG. 12A illustrates a top down view of a lower plate movably coupled to a rail in accordance with one embodiment of the present disclosure. FIG. 12B illustrates a side view of a lower plate movably coupled to a rail where the rail is horizontally offset by a predetermined amount in accordance with one embodiment of the present disclosure. FIG. 13A illustrates the relative positions in which a lower plate may be moved in accordance with one embodiment of the present disclosure. FIG. 13B illustrates the relative positions in which a lower plate may be moved in accordance with one embodiment of the present disclosure. FIG. 14A illustrates the progression of a lower plate from a first area, to a second area, and to a third area during processing in accordance with one embodiment of the present disclosure. FIG. 14B illustrates a cam mechanism, in communication with an upper plate, and wherein the cam mechanism exerts a pressure onto the upper plate (or a structure disposed therebetween) to exert or to maintain a sealing engagement between the upper and lower plates in accordance with embodiment of the present disclosure. FIG. 14C illustrates a cam mechanism, in communication with an upper plate, and wherein the cam mechanism is adapted to exert a force onto the upper plate (or a structure disposed therebetween) to exert or to maintain a sealing engagement between the upper and lower plates in accordance with embodiment of the present disclosure. FIG. 14D illustrates a side view of an assembly including a force generating member in accordance with one embodiment of the present disclosure. FIG. 14E illustrates a piston, in communication with an upper plate, and wherein the piston is adapted to exert a pressure onto the upper plate (or a structure disposed therebetween) to exert or to maintain a sealing engagement between the upper and lower plates in accordance with embodiment of the present disclosure. FIG. 14F illustrates a cam mechanism, in communication with an upper plate, and wherein the cam mechanism is adapted to exert a force onto the upper plate (or a structure disposed therebetween) to exert or to maintain a sealing engagement between the upper and lower plates in accordance with embodiment of the present disclosure. FIG. 14G illustrates a piston, in communication with an upper plate, and wherein the piston is adapted to exert a pressure onto the upper plate (or a structure disposed therebetween) to exert or to maintain a sealing engagement between the upper and lower plates in accordance with embodiment of the present disclosure. FIG. 14H illustrates a cam mechanism, in communication with an upper plate, and wherein the cam mechanism is adapted to exert a force onto the upper plate (or a structure disposed therebetween) to exert or to maintain a sealing engagement between the upper and lower plates in accordance with embodiment of the present disclosure. FIG. 14I illustrates multiple cam mechanisms joined together through a single cam shaft in accordance with one embodiment of the present disclosure. FIG. 14J illustrates a piston, in communication with an upper plate, and wherein the piston is adapted to exert a pressure onto the upper plate (or a structure disposed therebetween) to exert or to maintain a sealing engagement between the upper and lower plates in accordance with embodiment of the present disclosure. FIG. 14K illustrates a cam mechanism, in communication with an upper plate, and wherein the cam mechanism is adapted to exert a force onto the upper plate (or a structure disposed therebetween) to exert or to maintain a sealing engagement between the upper and lower plates in accordance with embodiment of the present disclosure. FIG. 14L illustrates a piston, in communication with an upper plate, and wherein the piston is adapted to exert a pressure onto the upper plate (or a structure disposed therebetween) to exert or to maintain a sealing engagement between the upper and lower plates in accordance with embodiment of the present disclosure. FIG. 14M illustrates a cam mechanism, in communication with an upper plate, and wherein the cam mechanism is adapted to exert a force onto the upper plate (or a structure disposed therebetween) to exert or to maintain a sealing engagement between the upper and lower plates in accordance with embodiment of the present disclosure. FIG. 15A provides a top view of an assembly including a vessel in which an unmasking operation may be performed in accordance with one embodiment. FIG. 15B provides a side view of an assembly including a vessel and a hinged upper plate in accordance with one embodiment of the present disclosure. FIG. 15C provides a perspective view of an assembly including a vessel in which an unmasking operation may be performed in accordance with one embodiment. FIG. 15D provides a perspective view of an assembly including a vessel in which an unmasking operation may be performed in accordance with one embodiment. FIG. 15E provides a perspective view of an assembly including a vessel in which an unmasking operation may be performed in accordance with one embodiment. FIG. 16A illustrates the progression of a lower plate from a first area, to a second area, and to a third area during processing in accordance with one embodiment of the present disclosure. FIG. 16B illustrates the progression of a lower plate having a wedge-based shape from a first area, to a second area, and to a third area during processing in accordance with one embodiment of the present disclosure. FIG. 16C illustrates the progression of a lower plate having a wedge-based shape from a first area, to a second area, and to a third area during processing in accordance with one embodiment of the present disclosure. FIG. 16D illustrates lower and upper plates having wedge-based shapes and where the upper and lower plates may be independently moved toward each other in accordance with one embodiment of the present disclosure. FIG. 17A illustrates a system having a variety of interoperable modules in accordance with one embodiment of the present disclosure. FIG. 17B illustrates a system having four discrete assemblies in accordance with one embodiment of the present disclosure. FIG. 17C illustrates a system having a plurality of discrete assemblies in accordance with one embodiment of the present disclosure. FIG. 18A illustrates an assembly in accordance with one embodiment of the present disclosure. FIG. 18B illustrates an assembly in accordance with one embodiment of the present disclosure. FIG. 18C illustrates an assembly in accordance with one embodiment of the present disclosure. FIG. 19A illustrates a top view of an assembly including a carousel, one or more substrate loaders, and a lower plate in accordance with one embodiment of the present disclosure. FIG. 19B illustrates a side view of the assembly of FIG. 19A including a carousel, one or more substrate loaders, and a lower plate in accordance with one embodiment of the present disclosure. FIG. 19C illustrates a perspective view of the assembly of FIG. 19A including a carousel, one or more substrate loaders, and a lower plate in accordance with one embodiment of the present disclosure. FIG. 20A illustrates a perspective view of a system including a plurality of carousels, substrate holders, substrate loaders, lower plates, and upper plates in accordance with one embodiment of the present disclosure. FIG. 20B illustrates a top view of a system including a plurality of carousels, substrate holders, substrate loaders, lower plates, and upper plates in accordance with one embodiment of the present disclosure. FIG. 21A illustrates the arrangement of heating and / or cooling elements in relation to an upper plate and a lower plate in accordance with one embodiment of the present disclosure. FIG. 21B illustrates the arrangement of heating and / or cooling elements in relation to an upper plate and a lower plate in accordance with one embodiment of the present disclosure. FIG. 21C illustrates the arrangement of heating and / or cooling elements in relation to an upper plate and a lower plate in accordance with one embodiment of the present disclosure. FIG. 21D illustrates the arrangement of heating and / or cooling elements in relation to an upper plate and a lower plate in accordance with one embodiment of the present disclosure. FIG. 22A illustrates a lower plate configured with two substrate stages in accordance with one embodiment of the present disclosure, where the lower plate is complementary to the upper plate of FIG. 22B. FIG. 22B illustrates an upper plate configured with two recessed cavities in accordance with one embodiment of the present disclosure, wherein the upper plate is complementary to the lower plate of FIG. 22A. FIG. 23A illustrates upper and lower plates in proximity to one another, wherein the upper plate includes a deformable sealing element and where the lower plate includes a tab in accordance with one embodiment of the present disclosure. FIG. 23B illustrates upper and lower plates in engagement with one another, wherein the upper plate includes a deformable sealing element and where the lower plate includes a tab in accordance with one embodiment of the present disclosure. FIG. 24A illustrates a side cross-sectional view of body of a lower plate including one or more heating and / or cooling elements in accordance with one embodiment of the present disclosure. FIG. 24B illustrates a side cross-sectional view of body of a lower plate including one or more heating and / or cooling elements in accordance with one embodiment of the present disclosure. FIG. 24C illustrates a side cross-sectional view of body (longitudinal side of the body) of a lower plate including one or more heating and / or cooling elements in accordance with one embodiment of the present disclosure. FIG. 24D illustrates a side cross-sectional view of body of a lower plate including one or more heating and / or cooling elements in accordance with one embodiment of the present disclosure. FIG. 25A illustrates a side cross-sectional view of body of a lower plate including one or more heating and / or cooling elements (e.g. bores into which one or more heating modules, e.g. heating cartridges, may be inserted) in accordance with one embodiment of the present disclosure. FIG. 25B illustrates a side cross-sectional view of body of a lower plate including one or more heating and / or cooling elements (e.g. one or more fluid channels serving as conduits for a fluid which may be heated and / or cooled, such as heated and / or cooled to a predetermined temperature or heated and / or cooled on-demand according to commands from a control unit) in accordance with one embodiment of the present disclosure. FIG. 25C illustrates a side cross-sectional view of body of a lower plate including one or more heating and / or cooling elements (e.g. bores for the insertion of one or more heating modules; fluid channels) in accordance with one embodiment of the present disclosure. FIG. 25D illustrates a side cross-sectional view of body of a lower plate including one or more heating and / or cooling elements (e.g. a plurality of fluid channels) in accordance with one embodiment of the present disclosure. FIG. 25E illustrates a side cross-sectional view of body of a lower plate including one or more heating and / or cooling elements (e.g. a thermoelectric module) in accordance with one embodiment of the present disclosure. FIG. 25F illustrates a top down cut-away view of body of a lower plate including one or more heating and / or cooling elements (e.g. bores for the insertion of one or more heating modules; fluid channels) in accordance with one embodiment of the present disclosure. FIG. 25G illustrates a top down cut-away view of body of a lower plate including one or more heating and / or cooling elements (e.g. a network of fluid channels) in accordance with one embodiment of the present disclosure. FIG. 25H illustrates a top down cut-away view of body of a lower plate including one or more heating and / or cooling elements (e.g. two separate networks of fluid channels) in accordance with one embodiment of the present disclosure. FIGS. 25I and 25J illustrate fluid flow through fluid channels in a lower plate in accordance with one embodiment of the present disclosure. FIG. 26A illustrates a side cross-sectional view of a body in communication with a thermal management module, wherein the thermal management module includes one or more heating and / or cooling elements in accordance with one embodiment of the present disclosure. FIG. 26B illustrates a side cross-sectional view of a body in communication with a thermal management module, wherein the body and the thermal management module both include one or more heating and / or cooling elements in accordance with one embodiment of the present disclosure. FIG. 26C illustrates a side cross-sectional view of a body in communication with a thermal management module, wherein the thermal management module includes one or more heating and / or cooling elements in accordance with one embodiment of the present disclosure. FIG. 26D illustrates a side cross-sectional view of a body in communication with a thermal management module, wherein the body and the thermal management module both include one or more heating and / or cooling elements in accordance with one embodiment of the present disclosure. FIG. 26E illustrates a side cross-sectional view of a body in communication with a thermal management module, wherein the body and the thermal management module both include one or more heating and / or cooling elements in accordance with one embodiment of the present disclosure. FIG. 26F illustrates a specimen processing apparatus including a substrate supported by body, the body being in thermal communication with a thermal management module in accordance with one embodiment of the present disclosure. FIG. 26G illustrates a specimen processing apparatus including a substrate supported by body, the body being in thermal communication with a thermal management module in accordance with one embodiment of the present disclosure. FIG. 27A illustrates a body set within a carrier in accordance with one embodiment of the present disclosure. FIG. 27B illustrates a perspective view of a body inserted or embedded within a carrier in accordance with one embodiment of the present disclosure. FIG. 27C illustrates a perspective view of a body inserted or embedded within a carrier in accordance with one embodiment of the present disclosure. FIG. 27D illustrates top view of a body inserted or embedded within a carrier in accordance with one embodiment of the present disclosure. FIG. 27E illustrates a side cut-away view of a body inserted or embedded within a carrier in accordance with one embodiment of the present disclosure. FIG. 27F illustrates a perspective view of a body inserted or embedded within a carrier, where the carrier is in communication with a pickup member in accordance with one embodiment of the present disclosure. FIG. 27G illustrates a perspective view of a body inserted or embedded within a carrier, where the carrier is in communication with a pickup member, and where the body and carrier are in thermal communication with a thermal management module in accordance with one embodiment of the present disclosure. FIG. 27H illustrates a perspective view of a body inserted or embedded within a carrier, where the carrier is in communication with a pickup member, and where the body and carrier are positioned over a thermal management module in accordance with one embodiment of the present disclosure. FIG. 27I illustrates a forklift device moving a carrier including a body to a thermal management module in accordance with one embodiment of the present disclosure. FIG. 28A illustrates a top down view of a body of a lower plate in accordance with one embodiment of the present disclosure. FIG. 28B illustrates a top down view of a body of a lower plate in accordance with one embodiment of the present disclosure. FIG. 28C illustrates a perspective view of a body of a lower plate including a vacuum port and / or one or more indentations in accordance with one embodiment of the present disclosure. FIG. 28D illustrates a body of a lower plate including one or more indentations in accordance with one embodiment of the present disclosure. The figure also depicts a gripper device positioned near a substrate. FIG. 28E illustrates a body of a lower plate including one or more indentations in accordance with one embodiment of the present disclosure. The figure also depicts a gripper device positioned near a substrate. FIG. 28F illustrates a body of a lower plate including one or more indentations in accordance with one embodiment of the present disclosure. The figure also depicts a gripper device positioned near a substrate. FIG. 29A illustrates a side cross-sectional view of body of an upper plate including one or more heating and / or cooling elements in accordance with one embodiment of the present disclosure. FIG. 29B illustrates a side cross-sectional view of body of an upper plate including one or more heating and / or cooling elements in accordance with one embodiment of the present disclosure. FIG. 29C illustrates a side cross-sectional view of body of an upper plate including one or more heating and / or cooling elements (e.g. fluid channels) in accordance with one embodiment of the present disclosure. FIG. 29D illustrates a side cross-sectional view of body of an upper plate including one or more heating and / or cooling elements (e.g. bore for the insertion of one or more heating modules; fluid channels) in accordance with one embodiment of the present disclosure. FIG. 29E illustrates a side cross-sectional view of body of an upper plate including one or more heating and / or cooling elements (e.g. bore for the insertion of one or more heating modules) in accordance with one embodiment of the present disclosure. FIG. 29F illustrates a top down cut-away view of body of an upper plate including one or more heating and / or cooling elements (e.g. bores for the insertion of one or more heating modules; fluid channels) in accordance with one embodiment of the present disclosure. FIG. 29G illustrates a top down cut-away view of body of an upper plate including one or more heating and / or cooling elements (e.g. a network of fluid channels) in accordance with one embodiment of the present disclosure. FIG. 30A illustrates a side cross-sectional view of a lower plate and an upper plate in communication with one another and where a chamber is formed therebetween. The figure also illustrates that the upper and / or lower plates include one or more heating and / or cooling elements in accordance with one embodiment of the present disclosure. FIG. 30B illustrates a side cross-sectional view of a lower plate and an upper plate in communication with one another and where a chamber is formed therebetween. The figure also illustrates that the upper and / or lower plates include one or more heating and / or cooling elements (fluid channels; bore for the insertion of one or more heating modules) in accordance with one embodiment of the present disclosure. FIG. 30C illustrates a side cross-sectional view of a lower plate and an upper plate in communication with one another and where a chamber is formed therebetween. The figure also illustrates that the upper and / or lower plates include one or more heating and / or cooling elements (fluid channels) in accordance with one embodiment of the present disclosure. FIG. 30D illustrates a side cross-sectional view of a lower plate and an upper plate in communication with one another and where a chamber is formed therebetween. The figure also illustrates that the upper and / or lower plates include one or more heating and / or cooling elements (fluid channels; thermoelectric modules) in accordance with one embodiment of the present disclosure. FIGS. 30E and 30F each illustrate a fluid flow path between upper and lower plates, each of the upper and lower plates including one or more fluid channels. FIG. 31A illustrates a specimen processing assembly including a lower plate movably coupled to a sub-assembly, wherein the lower plate includes a lower engagement surface and one or more substrate stages; an upper plate having an upper engagement surface complementary to the lower engagement surface; and a motorized vice in accordance with one embodiment of the present disclosure. FIG. 31B illustrates a lower plate including a lower engagement surface and one or more substrate stages; a sub-assembly for transporting the lower plate; an upper plate having an upper engagement surface complementary to the lower engagement surface; and a jack screw mechanism in accordance with one embodiment of the present disclosure. FIG. 31C illustrates a lower plate including a lower engagement surface and one or more substrate stages; a sub-assembly for transporting the lower plate; an upper plate having an upper engagement surface complementary to the lower engagement surface; and a scissor jack mechanism in accordance with one embodiment of the present disclosure. FIG. 31D illustrates a lower plate including a lower engagement surface and one or more substrate stages; a sub-assembly for transporting the lower plate; an upper plate having an upper engagement surface complementary to the lower engagement surface; and a scissor jack mechanism in accordance with one embodiment of the present disclosure. FIG. 31E illustrates a lower plate including a lower engagement surface and one or more substrate stages; a sub-assembly for transporting the lower plate; an upper plate having an upper engagement surface complementary to the lower engagement surface; and a scissor jack mechanism in accordance with one embodiment of the present disclosure. FIG. 32A illustrates a body having one or more attachment members in accordance with one embodiment of the present disclosure. FIG. 32B illustrates a body a roughened or patterned surface in accordance with one embodiment of the present disclosure. FIG. 32C illustrates a body having a coating disposed thereon in accordance with one embodiment of the present disclosure. FIG. 33A illustrates grippers arms of a gripper device in accordance with one embodiment of the present disclosure. FIG. 33B illustrates grippers arms of a gripper device in accordance with one embodiment of the present disclosure. FIG. 33C illustrates grippers arms of a gripper device in accordance with one embodiment of the present disclosure. FIG. 33D illustrates grippers arms of a gripper device in accordance with one embodiment of the present disclosure. FIG. 33E illustrates a gripper device including a suction cup in accordance with one embodiment of the present disclosure. FIGS. 34A illustrates the movement of a substrate with a gripper device to a specimen processing assembly, and the subsequent position of the substrate onto a body of a lower plate of the specimen processing assembly in accordance with one embodiment of the present disclosure. FIG. 34B illustrates the placement of a substrate onto a body of a lower plate using a gripper device and gripper arms in accordance with one embodiment of the present disclosure. FIG. 35A illustrates a forklift device in accordance with one embodiment of the present disclosure. FIG. 35B illustrates a forklift device in accordance with one embodiment of the present disclosure. FIG. 35C illustrates a forklift device in accordance with one embodiment of the present disclosure. FIG. 35D illustrates a forklift device in accordance with one embodiment of the present disclosure. FIG. 36A illustrates a forklift device including a cylinder which acts upon an upper surface of a substrate in accordance with one embodiment of the present disclosure. FIG. 36B illustrates an alternative view of a forklift device including a cylinder which acts upon an upper surface of a substrate in accordance with one embodiment of the present disclosure. FIG. 36C illustrates an alternative view of a forklift device including a cylinder which acts upon an upper surface of a substrate in accordance with one embodiment of the present disclosure. FIG. 36D illustrates an alternative view of a forklift device including a cylinder which acts upon an upper surface of a substrate in accordance with one embodiment of the present disclosure. FIG. 36E illustrates an alternative view of a forklift device including a cylinder which acts upon an upper surface of a substrate in accordance with one embodiment of the present disclosure. FIG. 36F illustrates the pick-up of a substrate using the forklift device depicted in FIGS. 36A to 36E. FIG. 37A illustrates a gripper device for picking up and moving a carrier in accordance with one embodiment of the present disclosure. FIG. 37B illustrates a gripper device for picking up and moving a carrier in accordance with one embodiment of the present disclosure. FIG. 37C illustrates a gripper device for picking up and moving a carrier in accordance with one embodiment of the present disclosure. FIG. 38A illustrates a substrate loading station having a platform and raised sides in accordance with one embodiment of the present disclosure. FIG. 38B illustrates a substrate loading station having a platform and raised sides in accordance with one embodiment of the present disclosure. FIG. 38C illustrates a substrate loading station having a platform and one or more spring mechanisms in accordance with one embodiment of the present disclosure. FIG. 38D illustrates a substrate loading station having a platform and one or more spring mechanisms in accordance with one embodiment of the present disclosure. FIG. 38E illustrates a substrate loading station having a platform and one or more alignment grippers in accordance with one embodiment of the present disclosure. FIG. 38F illustrates a substrate loading station having a platform and one or more alignment grippers in accordance with one embodiment of the present disclosure. FIG. 38G illustrates a substrate loading station having a platform and one or more alignment grippers in accordance with one embodiment of the present disclosure. FIG. 38H illustrates a substrate loading station having a platform and one or more rollers in accordance with one embodiment of the present disclosure. FIG. 39A illustrates an upper plate and a lower plate in communication with one another, thereby forming a chamber in accordance with one embodiment of the present disclosure. The figure further illustrates four different thermal zones which may be independently regulated, such that at least a portion of the substrate or the substrate stage remains, comparatively, the coldest component within the formed chamber. FIG. 39B illustrates an upper plate and a lower plate in communication with one another, thereby forming a chamber in accordance with one embodiment of the present disclosure. The figure further illustrates five different thermal zones which may be independently regulated, such that at least a portion of the substrate or the substrate stage remains, comparatively, the coldest component within the formed chamber. FIG. 39C illustrates an upper plate and a lower plate in communication with one another, thereby forming a chamber in accordance with one embodiment of the present disclosure. The figure further illustrates six different thermal zones which may be independently regulated, such that at least a portion of the substrate or the substrate stage remains, comparatively, the coldest component within the formed chamber. FIG. 40A illustrates a top-down view of a lower plate where different thermal zones A, B, B', C, and D are illustrated. In some embodiments, at least a portion of thermal zone A is in communication with a substrate, and wherein at least a portion of the substrate is maintained at a temperature which is less than the temperature in thermal zones B, B', C, and / or D. FIG. 40B illustrates a side cut-away view of an upper plate in communication with a lower plate, thereby forming a chamber in accordance with one embodiment of the present disclosure. The figure further illustrates thermal zones A, B, C, D, E, and F which may be independently regulated, such that at least a portion of a substrate disposed within the formed chamber remains at temperature which is less than the temperature of at least thermal zones C, D, E, and F. FIG. 40C illustrates a top-down view of an upper plate where different thermal zones E and F are illustrated. In some embodiments, at least a portion of thermal zone E is in communication with a substrate (such a substrate disposed within a chamber formed from the upper plate and a lower plate), and wherein at least a portion of the substrate is maintained at a temperature which is less than the temperature in thermal zones E and F. FIGS. 41A, 41B, and 41C each illustrate a thermal analysis of an upper plate and / or a lower plate in communication with one or more independently controllable heating and / or cooling elements. FIGS. 42A and 42B illustrate fluid channels in communication with a lower plate and other system components which enable the fluid within the fluid channels to be cooled to a pre-determined temperature. FIGS. 43A - 43H provide flow charts illustrating methods of performing an unmasking operation using any of the specimen processing assemblies and / or systems of the present disclosure. FIG. 44 sets forth an example of the times and temperatures used in a temperature ramp-up phase, a temperature maintenance phase, and a temperature ramp-down phase. FIG. 45 provides an example of the temperatures generated using a specimen processing assembly in accordance with one embodiment of the present disclosure. FIG. 46 provides a schematic 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

[0124] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.Definitions

[0125] As used herein, the singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "includes" is defined inclusively, such that "includes A or B" means including A, B, or A and B.

[0126] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, for example, the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (for example "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of" or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0127] The terms "comprising," "including," "having," and the like are used interchangeably and have the same meaning. Similarly, "s," "includes," "has," and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of "comprising" and is therefore interpreted to be an open term meaning "at least the following," and is also interpreted not to exclude 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. Moreover, while the steps and processes may be outlined herein in a particular order, the skilled artisan will recognize that the ordering steps and processes may vary.

[0128] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. 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 refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0129] As used herein, the term "antigen" refers to a substance to which an antibody, an antibody analog (e.g. an aptamer), or antibody fragment binds. Antigens may be endogenous whereby they are generated within the cell as a result of normal or abnormal cell metabolism, or because of viral or intracellular bacterial infections. Endogenous antigens include xenogenic (heterologous), autologous and idiotypic or allogenic (homologous) antigens. Antigens may also be tumor-specific antigens or presented by tumor cells. In this case, they are called tumor-specific antigens (TSAs) and, in general, result from a tumor-specific mutation. Antigens may also be tumor-associated antigens (TAAs), which are presented by tumor cells and normal cells. Antigen also includes CD antigens, which refers any of a number of cell-surface markers expressed by leukocytes and can be used to distinguish cell lineages or developmental stages. Such markers can be identified by specific monoclonal antibodies and are numbered by their cluster of differentiation.

[0130] As used herein, the term "fluid" refers to any liquid or liquid composition, including water, solvents, solutions (e.g. polar solvents, non-polar solvents), mixtures, colloids, suspensions, etc., used in a specimen processing operation that involves adding liquid or a composition including a liquid to a substrate, a specimen disposed on a substrate, etc. The fluid may be aqueous or nonaqueous. Non-limiting examples of fluids include solvents and / or solutions for deparaffinizing paraffin-embedded biological specimens, aqueous detergent solutions, washing solutions, rinsing solutions, acidic solutions, alkaline solutions, transfer solutions, and hydrocarbons (e.g., alkanes, isoalkanes and aromatic compounds such as xylene). Still further examples of fluids include solvents (and mixtures thereof) used to dehydrate or rehydrate biological specimens. In some embodiments, washing solutions include a surfactant to facilitate spreading of the washing liquids over the specimen-bearing surfaces of the slides. 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, transfer solutions include one or more glycol ethers, such as one or more propylene-based glycol ethers (e.g., propylene glycol ethers, di(propylene glycol) ethers, and tri(propylene glycol) ethers, ethylene-based glycol ethers (e.g., ethylene glycol ethers, di(ethylene glycol) ethers, and tri(ethylene glycol) ethers), and functional analogs thereof. Additional wash solutions, transfer solutions, acid solutions, and alkaline solutions, as well as their methods of application, and instruments for applying them are described in United States Patent Application Publication No. 2016 / 0282374.

[0131] As used herein, the "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 ranges of small angles from horizontal, for example, angles between about 0.1 degrees and 1.8 degrees from horizontal, such as angles between about 0.2 degrees and about 1.2 degrees, for example angles between about 0.3 degrees and about, 0.8 degrees. For example, a substrate that is positioned or held horizontal will have an orientation such that the large surfaces of the substrate are generally facing up and down, and which are substantially parallel to the ground. In particular embodiments, a rectangular substrate, such as a microscope slide, that is held horizontal will have an angle with respect to horizontal of between about 0.0 degrees and about 2.0 degrees along its short axis and an angle with respect to horizontal of between about 0.0 degrees and 2.0 degrees along its long axis, again with the large surfaces of the substrate generally facing up and down. Likewise, a sub-assembly (e.g. a rail) that is arranged or positioned horizontally is one that is substantially parallel to the ground or has an angle within about + / -2 degrees of horizontal.

[0132] As used herein, the term "plurality" refers to two or more, for example, 3 or more, 4 or more, 5 or more, etc.

[0133] As used herein, the terms "reagent" or "rare reagent" refer to solutions or suspensions including specific-binding entities, antibodies (primary antibodies, secondary antibodies, or antibody conjugates), nucleic acid probes (an isolated nucleic acid or an isolated synthetic oligonucleotide, attached to a detectable label or reporter molecule), unmasking agents (defined herein), detection probes, and solutions or suspensions of dye or stain molecules (e.g., H&E staining solutions, Pap staining solutions, etc.). The term "detection probes" refers to nucleic acid probes or antibodies which bind to specific targets (e.g. nucleic acid sequences, proteins, etc.). The detection probes may include a label for detection, such as radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens (including, but not limited to, DNP), and enzymes. Alternatively, the detection probes may contain no label or tag and may be detected indirectly (e.g. with a secondary antibody that is specific for the detection probe). The term "antibody conjugates" refers to those antibodies conjugated (either directly or indirectly) to one or more labels, where the antibody conjugate is specific to a particular target and where the label is capable of being detected (directly or indirectly). For example, an antibody conjugate may be coupled to a hapten such as through a polymeric linker and / or spacer, and the antibody conjugate, by means of the hapten, may be indirectly detected. As an alternative example, an antibody conjugate may be coupled to a fluorophore, such as through a polymeric linker and / or spacer, and the antibody conjugate may be detected directly. Examples of antibody conjugates are described in US Publication No. 2014 / 0147906 and U.S. Pat. Nos. 8,658,389; 8,686,122; 8,618,265; 8,846,320; and 8,445,191.

[0134] As used herein, the term "slide" refers to any substrate (e.g., substrates made, in whole or in part, glass, quartz, plastic, silicon, etc.) of any suitable dimensions on which a biological specimen is placed for analysis, and more particularly to a "microscope slide" such as a standard 3 inch by 1 inch microscope slide or a standard 75 mm by 25 mm microscope slide. Examples of biological specimens that can be placed on a slide include, without limitation, a cytological smear, a thin tissue section (such as from a biopsy), and an array of biological specimens, for example a tissue array, a cellular array, a DNA array, an RNA array, a protein array, or any combination thereof. Thus, in one embodiment, tissue sections, DNA samples, RNA samples, and / or proteins are placed on a slide at particular locations. In some embodiments, the term slide may refer to SELDI and MALDI chips, and silicon wafers.

[0135] As used herein, the term "specific binding entity" refers to a member of a specific-binding pair. Specific binding pairs are pairs of molecules that are characterized in that they bind each other to the substantial exclusion of binding to other molecules (for example, specific binding pairs can have a binding constant that is at least 10 3< M -1< greater, 10 4< M -1< greater or 10 5< M -1< greater than a binding constant for either of the two members of the binding pair with other molecules in a biological sample). Examples of specific binding moieties include specific binding proteins (for example, antibodies, lectins, avidins such as streptavidins, and protein A). Specific binding moieties can also include the molecules (or portions thereof) that are specifically bound by such specific binding proteins. Specific binding entities include primary antibodies, described above, or nucleic acid probes.

[0136] As used herein, the term "sample," "tissue sample," "specimen" or the like refers to any sample including a biomolecule (such as a protein, a peptide, a nucleic acid, a lipid, a carbohydrate, or a combination thereof) that is obtained from any organism including viruses. Other examples of organisms include mammals (such as humans; veterinary animals like cats, dogs, horses, cattle, and swine; and laboratory animals like mice, rats and primates), insects, annelids, arachnids, marsupials, reptiles, amphibians, bacteria, and fungi. Biological samples include tissue samples (such as tissue sections and needle biopsies of tissue), cell samples (such as cytological smears such as Pap smears or blood smears or samples of cells obtained by microdissection), or cell fractions, fragments or organelles (such as obtained by lysing cells and separating their components by centrifugation or otherwise). Other examples of biological samples include blood, serum, urine, semen, fecal matter, cerebrospinal fluid, interstitial fluid, mucous, tears, sweat, pus, biopsied tissue (for example, obtained by a surgical biopsy or a needle biopsy), nipple aspirates, cerumen, milk, vaginal fluid, saliva, swabs (such as buccal swabs), or any material containing biomolecules that is derived from a first biological sample. In certain embodiments, the term "biological sample" as used herein refers to a sample (such as a homogenized or liquefied sample) prepared from a tumor or a portion thereof obtained from a subject.

[0137] As used herein, the terms "stain," "staining," or the like as used herein generally refers to any treatment of a biological specimen that detects and / or differentiates the presence, location, and / or amount (such as concentration) of a particular molecule (such as a lipid, protein or nucleic acid) or particular structure (such as a normal or malignant cell, cytosol, nucleus, Golgi apparatus, or cytoskeleton) in the biological specimen. For example, staining can provide contrast between a particular molecule or a particular cellular structure and surrounding portions of a biological specimen, and the intensity of the staining can provide a measure of the amount of a particular molecule in the specimen. Staining can be used to aid in the viewing of molecules, cellular structures and organisms not only with bright-field microscopes, but also with other viewing tools, such as phase contrast microscopes, electron microscopes, and fluorescence microscopes. Some staining performed by the system can be used to visualize an outline of a cell. Other staining performed by the system may rely on certain cell components (such as molecules or structures) being stained without or with relatively little staining other cell components. Examples of types of staining methods performed by the system include, without limitation, histochemical methods, immunohistochemical methods, and other methods based on reactions between molecules (including non-covalent binding interactions), such as hybridization reactions between nucleic acid molecules. Staining methods include, but are not limited to, primary staining methods (e.g., H&E staining, Pap staining, etc.), enzyme-linked immunohistochemical methods, and in situ RNA and DNA hybridization methods, such as fluorescence in situ hybridization (FISH).

[0138] As used herein, the term "substantially" means the qualitative condition of exhibiting total or near-total 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%.

[0139] As used herein, the term "target" refers to any molecule for which the 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 bonded to proteins. Target molecules are typically detected using one or more conjugates of a specific binding molecule and a detectable label.

[0140] As used herein, the terms "unmask", or "unmasking" refer to retrieving antigens or targets and / or improving the detection of antigens, amino acids, peptides, proteins, nucleic acids, and / or other targets in fixed tissue. For example, it is believed that antigenic sites that can otherwise go undetected, for example, may be revealed by breaking some of the protein cross-links surrounding the antigen during the unmasking. In some embodiments, antigens and / or other targets are unmasked through the application of one or more unmasking agents (defined below), heat, and / or pressure. In some embodiments, only one or more unmasking agents are applied to the specimen to effectuate unmasking (for example no heating or pressure is required). In other embodiments, only heat is applied to effectuate unmasking. In some embodiments, unmasking may occur only in the presence of water and added heat. Unmasking is further described in United States Patent Publication Nos. 2009 / 0170152 and 2009 / 0104654. In some embodiments, unmasking may commence once an unmasking agent is applied to the specimen, regardless of when and where an unmasking operation takes place. For example, unmasking may start once an unmasking agent is applied to the specimen, but prior to commencing an unmasking operation.

[0141] As used herein, the term "unmasking operation" refers to the steps or phases of unmasking conducted within a sealed chamber. For example, an unmasking operation may include the steps of heating and / or pressurizing a specimen disposed on a substrate in the presence one or more unmasking agents (defined below) for a pre-determined amount of time in a sealed chamber. Other steps of an unmasking operation may include cooling the chamber or any portions thereof or depressurizing the sealed chamber. Yet other steps of an unmasking operation include quenching, flash boiling, and / or the step of dispensing additional fluids and / or reagents to the specimen while in the sealed chamber. In some embodiments, an unmasking operation may include a ramp-up phase, a temperature maintenance phase, a temperature ramp-down phase, a pressure ramp-up phrase, a pressure maintenance phase, and / or pressure ramp-down phase that occur after the chamber is formed.

[0142] As used herein, the term "unmasking agent" refers to any liquids, including solutions and mixtures, dispensed to a specimen to assist in unmasking. In some embodiments, the unmasking agent includes multiple components, such as the components recited below. In some embodiments, the unmasking agent is a buffer solution. In some embodiments, the buffer solution has a pH ranging from between about 5 and about 10. In other embodiments, the buffer solution has a pH ranging from between about 7 and about 9. In other embodiments, the buffer solution has a pH ranging from between about 7.5 and about 11. Non-liming examples of buffers include citric acid, potassium dihydrogen phosphate, boric acid, diethyl barbituric 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 unmasking agent is water. In other embodiments, the buffer solution may be comprised 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.

[0143] In some embodiments, the unmasking agent includes a TRIS-based buffer with a basic pH. In some embodiments, the TRIS-based buffer has a pH of about 10 (such as at elevated temperatures). In yet other embodiments, the buffer solution may be a citric acid sodium phosphate buffer solution having a pH of approximately 6.0 at elevated temperatures. In other embodiments, the unmasking agent includes about 0.05% citraconic anhydride. In other embodiments, the unmasking agent includes about 100 mM TRIS and has a pH of between about 8 and about 10. In other embodiments, the unmasking agent includes about 10 mM citrate, about 2 mM EDTA, and about 0.05% Tween 20, and has a pH of about 6.2. In other embodiments, the unmasking agent includes about 0.01M citrate buffer and has a pH of about 6.0.

[0144] In some embodiments, the unmasking agent includes a component which reacts with any liberated fixative to prevent it from reacting again with the sample. Examples a such unmasking agents include purpald or dimedone. Alternatively, or in addition, the unmasking agent can include a component that reacts in a reversible manner with free amino-groups of proteins and thus protects them from reaction with any available formaldehyde, e.g. citraconic anhydride (CCA).

[0145] In other embodiments, the unmasking agent includes a chelator. Examples of chelators 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'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN), citrate, or ionophores such as ionomycin or calcimycin, or any combination or mixtures thereof.

[0146] In other embodiments, the unmasking agent includes an enzyme. Non-limiting examples of enzymes include proteinases (such as trypsin, chymotrypsin, pepsin, or proteinase K), a nuclease, a glycanases, and a hyaluronidase.

[0147] In other embodiments, the unmasking agent includes a chaotropic agent. Non-limiting examples of chaotropic agents include butanol, ethanol, a guanidinium salt (for example, guanidinium hydrochloride or guanidinium thiocyanate), lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, thiourea and urea.

[0148] In other embodiments, the unmasking agent includes a nucleophile, for example a chemical species 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 halogenides.

[0149] In other embodiments, the unmasking agent includes 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.

[0150] In other embodiments, the unmasking agent can include a surfactant. As used herein, "surfactants" are classified as anionic, cationic, or nonionic, depending on their mode of chemical action. In general, surfactants reduce interfacial tension between two liquids. An example of a surfactant is sodium dodecyl sulfate. Examples of suitable nonionic surfactants include polyethylene glycol monohexadecyl ether, Cetostearyl alcohol, Cetyl alcohol, cocamide diethanolamine, cocamide monoethanolamine, Decyl glucoside, Octylphenoxypolyethoxyethanol, Polyethylene glycol monoisohexadecyl ether, Lauryl glucoside, Nonyl phenoxypolyethoxylethanol, 4-Nonylphenyl-polyethylene glycol, 1-(4-Nonylphenyl)-1,4,7,10,13,16,19,22,25-nonaoxaheptacosan-27-ol, nonoxynols, Monolaurin, Octaethylene glycol monododecyl ether, Oleyl alcohol, Polyethylene-polypropylene glycol, Polyglycerol polyricinoleate, Polysorbates, 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 names: Brij ®< 35, TWEEN ®< , Tergitol ™< , Triton ™< , Ecosurf ™< , Dowfax ™< , polysorbate 80 ™< , BigCHAP, Deoxy BigCHAP, IGEPAL ®< , Saponin, Thesit ®< , Nonidet ®< , Pluronic F-68, digitonin, deoxycholate, and the like. Particular disclosed working embodiments concern using surfactants selected from Brij ®< 35, TWEEN ®< , Tergitol ™< , Triton ™< . Additional antigen unmasking agents are disclosed in U.S. Patent No. 8,486,335.

[0151] In some embodiments, the unmasking agent includes deionized water, TRIS, and a chelator. In some embodiments, the unmasking agent includes deionized water, TRIS, and a chelator and has a pH ranging from between about 7 to about 9.5. In some embodiments, the unmasking agent includes deionized water, TRIS, a chelator, and a preservative. In some embodiments, the unmasking agent is CC1, available from Ventana Medical Systems, Inc., Tucson, AZ, USA. Further unmasking agents are further herein and described in United States Patent Publication No. 2009 / 0170152.OVERVIEW

[0152] As described herein, the present disclosure is directed to specimen processing assemblies including (a) a lower plate, and (b) an upper plate which is complementary to the lower plate. Upper and lower plates which are "complementary" each include features or sets of features which complement one another. For example, a lower plate may have a first set of features (e.g. a substrate stage and a lower engagement surface) and an 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 stage; and the upper engagement surface may be complementary to the lower engagement surface.

[0153] In some embodiments, the specimen processing assembly is configured such that the lower plate and the upper plate may be brought into contact with one another so as to form a chamber, such as a sealed chamber. In some embodiments, one or both of the lower and upper plates are movable. In some embodiments, the lower and upper plates are both independently movable, such as independently movable in any of the x, y, and z-coordinate directions. In other embodiments, one of the lower or the upper plates is movable, while the other of the lower or the upper plates is fixed. In some embodiments, the lower plate is moved toward a fixed upper plate. In other embodiments, the upper plate is moved toward a fixed lower plate.

[0154] In some embodiments, one or both of the lower and / or upper plates include one or more independently operable heating and / or cooling elements. In some embodiments, any of the heating and / or cooling elements within the upper plate may be operated in conjunction with those heating and / or cooling elements within the lower plate. As described further herein, any of the independently operable heating and / or cooling elements in the upper plate and / or lower plate may 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 a specimen (such as a specimen disposed on a substrate), a substrate, and / or the chamber may be controlled.

[0155] For example, it is possible to independently operate the various heating and / or cooling elements present in the upper and lower plates (or those in the thermal management modules) such that at least a portion of the substrate, or the specimen disposed on the substrate, remains the coldest structure within the chamber. For example, the substrate or the specimen disposed on the substrate may be maintained at a temperature lower than the upper plate, the lower plate, ports, valves, chamber walls, and / or any other structure within the chamber formed from the upper and lower plates.

[0156] In some embodiments, the one or more independently operable heating and / or cooling elements in any of the lower and / or upper plates may be arranged such that during their operation their arrangement and / or positioning allow for one or more temperature gradients to be established within the formed chamber (or along any constituent part 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 their operation a temperature gradient is established between the substrate stage and those portions of the chamber surrounding the substrate stage. In this regard, the temperature gradient enables the substrate stage (or at least a portion of the substrate disposed thereon) to be held at a comparatively lower temperature than other adjacent areas of the chamber. In some embodiments, a temperature gradient is established and maintained during all phases of an unmasking operation (e.g. heating, pressurizing, cooling, depressurization, quenching, etc.). In some embodiments, temperature gradients are established and maintained such that the specimen or a portion of the substrate are the "coldest" components within the formed chamber. For example, the specimen and / or the portion of the substrate may be maintained at a temperature which is less than the temperature of any other portions of the upper and / or lower plates, the chamber walls, nozzles, ports, temperature probes, pressure sensors, etc.

[0157] In some embodiments, the upper plate is coupled to one or more force generating members. In some embodiments, the one or more force generating members are selected from motors, screws, levers, pistons, cam mechanisms, etc. In some embodiments, the one or more force generating members exert a predetermined amount force onto at least the upper plate or any structure disposed therebetween. In some embodiments, the predetermined amount of force applied is less than a force generated by a predetermined threshold pressure within a chamber formed from the complementary upper and lower plates. In this regard, the predetermined force exerted by the force generating member is less than the forces pushing the upper and lower plates apart and, as a result, the force generating member may give way or slip such that pressure may be relieved from within the formed chamber. Alternatively, the predetermined amount of force applied does not exceed a force generated by a predetermined threshold pressure within the chamber. It is believed that this may act as a safety mechanism, thus mitigating the build-up of pressured within the chamber that exceed the predetermined threshold pressure. In some embodiments, the lower and / or upper plate may also include one or more ports and / or valves such that pressure which exceeds the predetermined threshold pressure may be released from the internal environment within the chamber. For example, a spring having a particular spring constant can be used as at least part of the force generating member such that when the internal pressure of the chamber exceeds a predetermined pressure, the spring will collapse further and release pressure from the chamber.

[0158] The present disclosure also is directed to specimen processing assemblies including a chamber, such as a chamber formed from a lower plate and an upper plate which is complementary to the lower plate. In some embodiments, unmasking (e.g. antigen retrieval, target retrieval) may be performed within the chamber. For example, gas and / or steam may be introduced into the chamber to pressurize the chamber. By way of another example, one or more heating elements may be activated to heat one or more fluids and / or reagents present within 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 a substrate, the specimen disposed on the substrate, or the chamber itself. In some embodiments, the chamber may have any size and / or shape. In some embodiments, the chamber is sized to accommodate at least a portion of a substrate and any specimens, fluids, and / or reagents disposed thereon.

[0159] The present disclosure is also directed to systems including one or more specimen processing assemblies. In some embodiments, the systems include one or more specimen processing assemblies and at least one dispense device. In some embodiments, the system further includes one or more liquid removal modules, mixing modules, pressurization modules, imaging modules, coverslipping modules, etc. In some embodiments, the systems include a control system having one or more processors and one or more memories.SPECIMEN PROCESSING ASSEMBLY COMPONENTS

[0160] As noted above, the specimen processing assemblies may include a lower plate, and an upper plate which is complementary to the lower plate. In some embodiments, the specimen processing assemblies further include at least one additional component, e.g. a sub-assembly, a support member, a substrate loader, a substrate holder, a force generating member, etc. Each of these components will be described further herein.LOWER PLATE

[0161] The specimen processing assemblies of the present disclosure include at least one lower plate, such as those illustrated in FIGS. 1 - 4, or a modular lower plate, such as those illustrated in FIGS. 26A - E.

[0162] With references to FIGS. 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, such as a microscope slide.

[0163] In some embodiments, the one or more substrate stages 12 are raised relative to the lower engagement surface 11. In other embodiments, the one or more substrate stages 12 are recessed relative to the lower engagement surface 11. In yet other embodiments, the one or more substrate stages 12 includes a first portion which is raised or level with the lower engagement surface 11 and a second portion which is recessed or level with the lower engagement surface 11.

[0164] In some embodiments, the substrate stage 12 has a shape which is substantially rectangular (see, e.g. FIG. 1A or FIG. 1D). In other embodiments, the substrate stage 12 has rounded edges. In yet other embodiments, the substrate stage 12 has chamfered edges 172 (see, e.g., FIG. 1H). In some embodiments, the substrate stage 12 approximates the size of a substrate. In other embodiments, the substrate stage 12 is larger than the size of a substrate. In yet other embodiments, the substrate stage 12 is smaller than the size of a substrate. For example, the size of a substrate stage 12 may be smaller than a standard microscope slide in at least one dimension. In some embodiments, a substrate stage which is sized smaller than the substrate allows for any condensation formed within a chamber formed from the lower plate and a complementary upper plate to form under the slide or migrate under the slide. In some embodiments, at least a portion of one edge of the substrate overhangs one side of the substrate stage.

[0165] In some embodiments, and as described herein, the lower plate 10 is configured to be complementary to an upper plate 30 (see, e.g., FIG. 6B). In some embodiments, the lower plate 10 is adapted such that a substrate 15 supported by substrate stage 12 remains horizontal while the lower plate 10 is moved. In this way, any fluids and / or reagents disposed thereon remain on the substrate, for example they do not migrate to the edges of the substrate or run off the substrate. In some embodiments, the body 16 of the lower plate 10 is monolithic. In other embodiments, the body 16 includes of two or more members coupled together. These and other features of the lower plate 10 are described herein.

[0166] In some embodiments, the body 16 of the lower plate 10 may have any size or shape, e.g. a polygonal shape or a wedge-based shape. Likewise, the lower engagement surface 11 may have any size or shape, provided that it is complementary to an upper engagement surface 31 of an upper plate 30 as described herein (see, e.g., FIG. 9A). In some embodiments, the lower engagement surface 11 of the lower plate 10 may have the same general shape as the lower plate. While not depicted, in some embodiments, the lower plate 10 may have a lower engagement surface that is not linear, but curvilinear, again provided that any upper engagement surface of an upper plate is complementary to the curvilinear lower engagement surface.

[0167] In some embodiments, and as depicted in any of FIGS. 1, 2, 3, and 4, the body 16 of the lower plate 10 may have a generally rectangular shape. In other embodiments, and with reference to FIG. 1A, the body 16 of the lower plate 10 includes a length 2 and width 3 whose general proportions conform to those of a microscope slide. In embodiments where the body 16 has a rectangular shape, a plane formed by the upper planar surface 12C of the substrate stage 12 is parallel with a plane formed by the lower engagement surface 11.

[0168] In other embodiments, the body 16 of the lower plate 10 has a wedge-based shape. For example, and as depicted in at least FIG. 6A, a side of body 16 may have a wedge-based shape which tapers from about a first height 4 to about a second height 5. FIGS. 6C and 6D show front-on end views, respectively, of the two ends of a wedge-based body 16. In some embodiments, a body 16 having a wedge-based shape, may further include a lower engagement surface 11 and a substrate stage 12 (FIG. 6A). In some embodiments, and as illustrated in FIG. 6A, 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) would intersect 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 the lower plate, coupled to a sub-assembly as described herein, traverses the sub-assembly.

[0169] With reference to FIGS. 6A and 6B, in some embodiments, a substrate stage first end 12A may be raised relative to a lower engagement surface first portion 11A; while a substrate stage second end 12B may be even with or recessed relative to a lower engagement surface second portion 11B. In some embodiments, and with reference to FIG. 6A, a substrate stage upper surface 12C is provided at a height 6 that is the same, higher, or lower than height 4. FIG. 6E provides an alternate wedge-based body 16 where a substrate stage first end 12A is raised relative to a lower engagement surface first portion 11A; while a substrate stage second end 12B is recessed relative to a lower engagement surface second portion 11B. FIG. 6F provides yet a further alternative lower plate 10 having a wedge-based body 16.

[0170] While not depicted in FIG. 6A, the substrate stage 12 may include one or more alignment members, such as described further herein. In some embodiments, the one or more alignment members have a height which is less than a height of a substrate disposed on a substrate stage. In other embodiments, the one or more alignment members have a height which is about half the height of the substrate, or less than half the height of the substrate. Additionally, the body 16 of the wedge-based lower plate 10 may include a groove, such as a groove circumscribing a periphery of a substrate stage 12. In some embodiments, the groove is adapted to at least partially engage a seal body as described further herein.

[0171] 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 depicted in FIGS. 1D through 1H. In some embodiments, the vacuum port 170 is plumbed through the body 16 and is in fluidic 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-round shape. In some embodiments, the one or more vacuum sealing members 171 are comprised of a material such as rubber, silicon, or a fluorocarbon rubber. In some embodiments, the one or more vacuum sealing members 171 comprise an O-ring. In some embodiments, the substrate stage includes 1 vacuum sealing member. In other embodiments, the substrate stage includes 2 or more vacuum sealing members. In yet other embodiments, the substrate stage includes 3 or more vacuum sealing members. In some embodiments, each of the one or more vacuum sealing members are contiguous with each other.

[0172] In some embodiments, the one or more vacuum sealing members 171 are compressible and / or temporarily deformable (e.g. compressible by about 5%, compressible by about 10%, compressible by about 15%, compressible by about 20%, compressible by about 25%, etc.). In some embodiments, the one or more vacuum sealing members 171 may be in an uncompressed state where 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 may be in a compressed state where the one or more vacuum sealing members 171 can be configured to maintain an airtight seal with a backside of a substrate (e.g. the backside of a microscope slide) such that the substrate is pulled against the surface 12C of the substrate stage 12 by a vacuum drawn via the one vacuum port 170.

[0173] In some embodiments, a vacuum may be drawn between the substrate and the substrate stage. For example, a sufficient vacuum may be drawn to inhibit or limit movement of the substrate along the substrate stage. The vacuum can be reduced or eliminated to remove the substrate from the substrate stage. Any of the vacuum ports, sealing members, sealing surfaces, vacuum lines, and vacuum sources described in United States Patent No. 9,989,448 may be utilized within the present disclosure. In addition, any of the methods of operation of the vacuum ports as described within United States Patent No. 9,989,448 may be adapted for use in the lower plates 10 of the present disclosure.

[0174] With reference to at least FIGS. 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 a substrate into an appropriate position on the upper surface 12C of the substrate stage 12, e.g. a position that is centered within the upper surface 12C of the substrate stage 12. In some embodiments, the substrate stage 12 may include between 1 and 10 alignment members. In some embodiments, the alignment members 14 are protuberances which project vertically from the surface of the substrate stage 12. In some embodiments, the protuberances may have any size and shape, such as cylindrical or polygonal. In some embodiments, the alignment members 14 are pins that project from the upper surface 12C of the substrate stage 12. In some embodiments, one or more alignment members 14 are located near one or both of the substrate stage ends 12A and 12B. In some embodiments, the one or more alignment members have a height which is less than the height of the substrate. In other embodiments, the one or more alignment members have a height which is about half the height of the substrate, or less than half the height of the substrate.

[0175] In the context of a microscope slide being supported by substrate stage 12, one or more alignment members 14 may be located on the substrate stage 12 such that they are positioned at the terminal ends of the microscope slide. For example, 1, 2, or 3 alignment members 14 may be positioned on the substrate stage 12 at a label end 15A of a microscope slide 15; and another 1, 2, or 3 alignment members 14 may be positioned on the substrate stage 12 at the opposing terminal end of the slide 15 (for example the specimen-bearing end). In some embodiments, one or more alignment members 14 may be located along the periphery of the longitudinal length of the substrate stage 12. By way of another example, and again in the context of a microscope slide, one or more alignment members 14 may 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 may be equally spaced apart from one another. In other embodiments, the alignment members 14 may be spaced randomly.

[0176] With reference to at least FIGS. 3A and 3B, in some embodiments, the lower plate 10 may further include a groove 13 which at least partially circumscribes the body 16 of the lower plate 10. As shown in FIGS. 3A and 4A, in some embodiments, the groove 13 may circumscribe 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 seal body may removably engage the groove walls (not depicted). In some embodiments, a seal body inserted into groove 13 may have a first portion which at least partially engages the groove walls; and a second portion which protrudes from the groove 13 and extends at least partially above engagement surfaces 11A and / or 11B (such that it is available for communication with an upper engagement surface 31). In some embodiments, the seal body is in the form of an O-ring seal. By an "O-ring seal" it is meant an annular seal, irrespective of the shape of its cross section. In some embodiments, the substrate stage 12 may further include one or more alignment members, as shown in FIGS. 4A - 4C.

[0177] In some embodiments, a seal body may be comprised of a material that is resistant to chemicals and / or heat. In some embodiments, the seal body is comprised of a metal. In some embodiments, the seal body is compressible and / or temporarily deformable. In some embodiments, the seal body is comprised of a material such as rubber, silicon, or a 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 rubbers include, but are not limited to, a vinylidene fluoride rubber of a binary system such as a vinylidene fluoride / hexafluoropropylene copolymer, a vinylidene fluoride / trifluorochloroethylene copolymer, and a vinylidene fluoride / pentafluoropropylene copolymer, a vinylidene fluoride rubber of a ternary system such as a vinylidene fluoride / tetrafluoroethylene / hexafluoropropylene copolymer, a vinylidene fluoride / tetrafluoroethylene / perfluoroalkylvinyl ether copolymer, and a vinylidene fluoride / tetrafluoroethylene / propylene copolymer, a tetrafluoroethylene / propylene copolymer, a tetrafluoroethylene / perfluoroalkylvinyl ether copolymer, and a thermoplastic fluorocarbon rubber.

[0178] In other embodiments, the seal body may be comprised of a perfluoroelastomer, such as those recited in US Patent No. 7,834,096. In yet other embodiments, the seal body may be comprised of nitriles, such as acrylonitrile-butadiene (NBR), hydrogenated nitrile (HNBR), and carboxylated nitrile (XNBR); fluorocarbon (FKM); fluorosilicone (FVMQ); perfluoroelastomer (FFKM); tetrafluoroethylene-propylene (FEPM); ethylene acrylate (AEM); polyacrylate; and thermoplastic polyurethane. In further embodiments, the seal may be comprised of a material that is heat-resistant, such as any of those described within US Patent No. 7,919,554.

[0179] In some embodiments, a seal body 18 is integrated within a removable seal attachment 17, such as depicted in FIG. 5B. In some embodiments, the removable seal attachment 17 is configured to engage a portion of the lower plate 10 (although, the removable seal attachment 17 could also be configured to engage a portion of the upper plate 30). For example, the removable seal attachment 17 may include one or more clips 24 which facilitate its attachment to a portion of the lower plate 10 (see, e.g., FIG. 5B). In other embodiments, the removable seal attachment 17 may frictionally engage a periphery of the lower plate 10. In yet other embodiments, the removable seal attachment 17 may be screwed into the lower plate 10. In some embodiments, the seal body 18 of the removable seal attachment 17 fits into groove 13 of the lower body and at least partially engages the walls of groove 13. In some embodiments, at least a portion of seal body 18 of the removable seal attachment 17 extends above an upper surface of the removable seal attachment 17. In some embodiments, the seal body is positioned such that it may contact a portion of an upper engagement surface 31. In some embodiments, the removable seal attachment 17 includes an upper surface 25 which acts as an engagement surface, such as a lower engagement surface for contacting and / or facilitating the formation of a seal with an upper engagement surface 31 of an upper plate 30. In some embodiments, the removable seal attachment 17 helps to retain the seal formed between the upper and lower plates, especially when a chamber formed from the upper and lower plates is pressurized. In some embodiments, the removable seal attachment 17 includes a cavity 26 such that the substrate stage 12 (and any substrate 15 supported by the substrate stage 12) may be exposed for further processing when the removable seal attachment 17 is coupled to the lower plate 10.

[0180] In some embodiments, the lower plate 10 includes one or more indentations within the body 16 and adjacent to the substrate stage 12. For example, FIGS. 28A, 28B, and 28C each illustrate a body 16 having two indentations 173A and 173B, where the one or more indentations are adjacent to both longitudinal sides of a substrate stage 12. The indentations 173A and 173B may be of any size and / or shape. Additionally, the indentations 173A and 173B may have any depth within the body 16. In some embodiments, the two indentations 173A and 173B are in alignment 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 stage 12 itself.

[0181] In some embodiments, the one or more indentations are sized to accommodate a gripper device, e.g. sized to accommodate the arms of a gripper device adapted to pick-up and move a substrate. Suitable gripper devices for picking up a slide supported by a substrate stage are described herein. FIGS. 28D and 28E illustrate a body 16 of a 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 illustrated in FIG. 28E, the gripper arms 461A and 461B are in an extended configuration, but that the gripper arms 461A and 461B may be retracted such that the gripper arms 461A and 461B move in the illustrated directions X and X'. Upon retraction of the gripper arms 461A and 461B, the notched ends 462A and 462B of the gripper 460 move towards the substrate 15 and at least partially surround the edges of the substrate 15, allowing it to be picked up by the gripper device 460 and moved, such as in the illustrated Z direction. In some embodiments, the indentations 173A and 173B within the body 16 are sized to accommodate at least the notched ends 462A and 462B in both the extended and retracted positions of gripper arms 461A and 461B.

[0182] 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, FIGS. 24A and 24C each depict a lower plate 10 having a body 16, whereby one or more heating and / or cooling elements 21 are shown 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, e.g. 1 or more heating and / or cooling elements, 2 or more heating and / or cooling elements, 3 or more heating and / or cooling elements, 4 or more heating and / or cooling elements, 5 or more heating and / or cooling elements, 6 or more heating and / or cooling elements, etc. Likewise, the body 16 of the lower plate 10 may include any combination of heating and / or cooling elements, e.g. 1 heating and / or cooling element; 1 heating 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.

[0183] In some embodiments, the one or more heating and / or cooling elements 21 may 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 parallel to one or more cooling elements 21B. The skilled artisan will appreciate, however, that the one or more heating and / or cooling elements may be arranged side-by-side (e.g. heating elements 401A and 401B of FIG. 25A), in a staggered arrangement (e.g., compare heating elements 401A and 401B versus heating elements 411A and 411B), may be spaced evenly, may be spaced randomly, etc. In some embodiments, one or more heating and / or cooling elements may be positioned beneath a substrate stage. In other embodiments, one or more heating and / or cooling elements may be positioned beneath a substrate stage while additional heating and / or cooling elements are positioned beside the one or more heating and / or cooling elements positioned beneath the substrate stage, whereby the additional heating and / or cooling elements flank the one or more heating and / or cooling elements positioned beneath the substrate stage (see FIGS. 25C, 25D, and 25F).

[0184] In some embodiments, the one or more heating and / or cooling elements may be arranged such that during their operation (such as during any heating and / or cooling steps, temperature ramp-up or temperature ramp-down steps), their arrangement and / or positioning relative to the body of the lower plate enables a temperature (or thermal) gradient to be established between different portions of the 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 stage and those portions of the body surrounding the substrate stage. In this regard, the temperature gradient enables the substrate stage (or at least a portion of the substrate disposed thereon) to be held at a comparatively lower temperature than other adjacent areas of the body 16 of the lower plate 10. For instance, and with reference to FIG. 40A, the surface 12C of substrate stage 12 may be maintained at a temperature A, while those portions of the body 16 of the lower plate 10 surrounding or adjacent to the substrate stage 12 may be maintained at temperatures B, B', C, and D. Following this particular example, a temperature gradient 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 less than any of the temperatures B, B', C, and D, the surface 12C of the substrate stage 12 will comparatively be the "coldest" portion of the lower plate 10.

[0185] 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 initiates, the temperature of the substrate stage is maintained at a temperature which is comparatively colder than the surrounding and / or adjacent portions of body 16. It is believed that establishing and maintaining the temperature gradient even during temperature ramp-up and ramp-down enables at least a portion of the substrate to be maintained comparatively cooler than other parts of the body of the lower plate. In this regard, when the lower plate and upper plate (described herein) are in communication with one another such that a chamber is formed therebetween (such as forming a chamber enclosing the substrate and / or substrate stage), at least a portion of the substrate and / or substrate stage are maintained as the "coldest" component within the formed chamber, for example, colder than any other portions of the upper and / or lower plate, colder than any nozzles, ports, temperature probes, pressure sensors, etc.

[0186] For example, the one or more heating and / or cooling elements may be independently operated such that during any temperature ramp-up phase, the surface 12C of substrate stage 12 is maintained at all times at a temperature which is less than the temperature of the surrounding and / or adjacent areas of body 16. That is, at any one particular time during a temperature ramp-up phase, the substrate stage 12 is maintained, in some embodiments, at a temperature lower than the surrounding and / or adjacent portions of body 16. For example, and with reference to FIG. 40A, assuming that a temperature ramp-up takes place over a 10-minute time period, if one were to take a temperature measurement at a time of 3 minutes, temperature A would be comparatively less than temperatures B, B', C, and / or D. Likewise, if one were to take a second temperature measurement at a time of 7 minutes, temperature A would be comparatively less than temperatures B, B', C, and / or D. Following this example further, once a pre-determined temperature is reached for any one of the portions A, B, B', C, and / or D (such as at a time point of 10 minutes), the temperature measurement of A taken at this time point would again be comparatively less than temperatures B, B', C, and / or D.

[0187] As described in further detail herein, this is believed to enable any substrate supported by the substrate stage (or any portion of the substrate) to have a comparatively lower temperature than adjacent portions of the body of the lower plate. In some embodiments, the temperature gradient between a substrate stage and portions of the body 16 flanking the substrate stage ranges from between 5°C and 50°C. In other embodiments, the temperature gradient between a substrate stage and portions of the body 16 flanking the substrate stage ranges from between 5°C and 40°C. In yet other embodiments, the thermal gradient between a substrate stage and portions of the body 16 flanking the substrate stage ranges from between 5°C and 30°C. In further embodiments, the temperature gradient between a substrate stage and portions of the body 16 flanking the substrate stage ranges from between 5°C and 25°C. In yet further embodiments, the temperature gradient between a substrate stage and portions of the body 16 flanking the substrate stage ranges from between 5°C and 20°C. In other embodiments, the temperature gradient between a substrate stage and portions of the body 16 flanking the substrate stage ranges from between 5°C and 15°C. In other embodiments, the temperature gradient between a substrate stage and portions of the body 16 flanking the substrate stage ranges from between 5°C and 10°C.

[0188] The one or more heating and / or cooling elements embedded within the body 16 of the lower plate 10 may be of any type known to those of skilled in the art, including any of those described herein. The skilled artisan will appreciate that in those lower plate embodiments which include more than one heating and / or cooling element, the same or different types of heating and / cooling elements may be utilized. By way of example only, the body 16 of lower plate 10 may include one or more fluid channels for circulating a fluid to effectuate heating and / or cooling of the body, and the body 16 may also include one or more bores into which one or more heating modules (e.g. heating cartridges) may be inserted. By way of another example, the body 16 of the lower plate 10 may include a thermoelectric cooling module to enable cooling to a temperature below room temperature and also one or more fluid tubes or channels for circulating a fluid having a temperature above room temperature.

[0189] In some embodiments, the one or more heating elements 21 include 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 includes a heating foil bent around a cylindrical inner core, such as a ceramic inner core, fully covered by a metallic cylinder.

[0190] The body 16 of lower plate 10 may include one or more bores into which a heating module, e.g. a heating cartridge may be inserted. In some embodiments, any number of bores may be provided within the body 16 of a lower plate 10, e.g. 1 bore, 2 bores, 3 bores, 4 bores, 6 bores, 8 bores, 12 bores, etc. The bores may be arranged in any manner within the body 16 of the lower plate. As such, the body 16 may accommodate 1, 2, 3, 4, 6, 8, 12, etc. heating cartridges. For example, FIGS. 25A and 25C each illustrates a body 16 having two bores 401A and 401B into which heating cartridges may be inserted.

[0191] In some embodiments, one or more bores may be evenly spaced apart from one another. In other embodiments, the one or more bores may be randomly spaced. In some embodiments, the bores are open to the longitudinal side of the lower plate 10 (see, e.g., FIG. 1D which illustrates the longitudinal side of the lower plate 10). Alternatively, in in other embodiments, the bores may be open on the "short side" of the lower plate, as illustrated in FIG. 25A. As illustrated in FIG. 25F, the bores may traverse the entire length of the body 16 of the lower plate (see bore 401B) or may run 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.

[0192] In some embodiments, each of the one or more heating cartridges inserted into the body 16 may be independently operable. For example, assuming that a body 16 of a lower plate 10 includes three heating cartridges inserted into each of three different bores, each of the three heating cartridges may be independently operable, for example heating cartridges 1 and 3 may be operated at 80% of their power rating while cartridge 2 is operated at 40% of its power rating.

[0193] In other embodiments, the one or more heating elements 21 embedded within the body 16 include a ceramic element 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 within the body 16 include a resistive electrical heater. In yet other embodiments, the one or more heating elements 21 embedded within the body 16 includes an induction coil. In further embodiments, the one or more heating elements embedded within the body 16 utilize one of infrared radiation or microwave radiation to heat a substrate or a sample disposed on the substrate. In some embodiments, infrared radiation or microwave radiation is used to heat a fluid, such as fluid present within a reservoir or present on a substrate. In these embodiments, the fluid is heated with infrared radiation or microwave radiation such that steam is generated. In some embodiments, the generated steam is used to heat the sample and / or pressurize a chamber formed from the lower plate and a complementary upper plate as described further herein.

[0194] 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 band. Additional embodiments illustrating the use of heating elements and / or cooling elements in the upper and lower plates are illustrate din FIGS. 21A -21D. In some embodiments, any of the heating and / or cooling elements within the lower plate 10 may be operated in conjunction with those heating and / or cooling elements within an upper plate 30 or with any thermal management module. As a result, the skilled artisan will appreciate that by controlling the various heating and / or cooling elements present in the upper and lower plates and / or in the thermal management module, that the temperature of the substrate stage, any substrate supported by the substrate stage, and / or the chamber may be regulated, e.g. regulated to maintain at least a portion of the specimen or at least a portion of the substrate as the coldest component within any chamber formed from upper and lower plates.

[0195] In some embodiments, the one or more heating elements or cooling elements 21 embedded within the body 16 includes a Peltier device or includes a thermoelectric module 402. Suitable Peltier devices include any of those described within U.S. Pat. Nos. 4,685,081, 5,028,988, 5,040,381, and 5,079,618. By way of example, FIG. 25E which illustrates a thermoelectric module embedded within the body 16 of the lower plate 10, where a "hot side" of the thermoelectric module is in thermal communication with the body 16 of the lower plate 10. Of course, a "cold side" of the thermoelectric module may similarly be in thermal communication with the body 16 of the lower plate 10.

[0196] In other embodiments, the one or more heating elements and / or cooling elements 21 embedded within the body 16 include one or more fluid channels, where the one or more fluid channels serve to circulate a fluid within the body 16 of the lower plate 10. In some embodiments, the fluid is a 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).

[0197] The one or more fluid channels embedded within the body 16 of the lower plate may have any configuration and may be independently operable. For example, and with reference to FIGS. 25B and 25F, the one or more fluid channels 411A and 411B may be arranged as two independent parallel conduits which independently permit the flow of a suitable fluid through the body 16, for example flow through a first opening (414A and 414B) on one side of the body 16, flow through the fluid channels 411A and 411B, and flow out of a second opening (413A and 413B) on the opposite side of body 16. In this particular example, the fluid channels 411A and 411B may each be configured for cooling, may each be configured for heating, or one may be configured for heating while the other is configured for cooling. Likewise, the fluid channels 411A and 411B, while both configured for heating, may be configured to heat at different temperatures.

[0198] FIG. 25D, which shows a cross section of a body 16, illustrates yet another example of a body 16 of a lower plate 10 including a plurality of fluid channels 411A - 411D. In this particular embodiment, the plurality of fluid channels 411A - 411D may each be independent fluid channels, may all be interconnected, or some of the fluid channels may be interconnected (e.g. 411B and 411C may be interconnected) while others are not. In some embodiments, some of the fluid channels 411A - 411D may be used for circulating a fluid for heating the body 16, the substrate stage 12, the substrate 15, or any specimen deposited on substrate 15; while others of the fluid channels 411A - 411D may be used for circulating a fluid for cooling the body 16, the substrate stage 12, the substrate 15, or any specimen deposited on substrate 15. In some embodiments, the one or more fluid channels are in communication with one or more fluid delivery line, ports, fluid reservoirs, heating devices, cooling devices, heat exchanges, pumps, and / or valves such that fluid may be supplied independently to each fluid channel.

[0199] Alternatively, the one or more fluids channels may include a network 412 of fluid channels, such as those depicted in FIG. 25G. The network 412 of fluid channels may include one or more independently controllable inlets 414, outlets 413, or valves (not depicted) so as to direct the circulation along a particular fluid flow path, or to provider circulation within only a portion of the network of channels. In some embodiments, the network of fluid channels may be in communication with a fluid reservoir (not depicted), and 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 including 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 may be independently operated, e.g. may each include a fluid heated to different temperatures, may each have different fluid flow rates, etc. FIGS. 25I and 25J both illustrate a lower plate 10 having at least one fluid channel 411 disposed therein, and wherein the fluid channel is in communication with a recirculation pump 601 and a chiller 602. In some embodiments, the fluid channel 411 is in further communication with a fluid reservoir.

[0200] In some embodiments, the substrate or the sample disposed on the substrate may be heated with steam, such as described herein. In these embodiments, heating and / or cooling elements within the lower plate may be used for thermal management in conjunction with the introduction of steam to a specimen disposed on a substrate.

[0201] 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. By way of example, an active cooling element may include one or more fluid channels to facilitate the flow of a coolant and thus provide active cooling of the sample, the upper plate, and / or the lower plate as noted above.

[0202] In other embodiments, the one or more cooling elements are passive cooling elements, e.g. 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 engageable with the body 16, such as described further herein. For example, as depicted in FIGS. 8A and 8B, a heat sink 29 may be integral with a housing member 20. In other embodiments, a heat sink may be fixedly secured to a bottom surface lower plate 10, e.g. may be glued onto, screwed onto, or clipped onto either 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.

[0203] As noted above, in those embodiments where the body 16 of 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, FIGS. 25C and 25F each illustrates a body 16 of a lower plate 10 including two different types of heating and / or cooling elements. In these specific example, the body 16 includes two fluid channels 411A and 411B (which may be interconnected and / or independent of one another) and two bores 401A and 401B into which heating cartridges may be inserted. In this particular embodiment, the two fluid channels may independently be used for heating and / or cooling the body 16, the substrate stage 12, the substrate 15, or any specimen deposited on substrate 15.

[0204] In some embodiments, the two independently operable fluid channels 411A and 411B may be used to establish a first temperature (e.g. 95°), while the two independently operable heating cartridges within bores 401A and 401B may be used to establish a second temperature (e.g. 140°C). In this way, a temperature gradient may be established across portions of the body 16 of the lower plate 10. Following the above example further, the portion of the body 16 beneath the substrate stage 12 may comparatively have a lower temperature than the temperature established at the periphery of the body 16 due to the arrangement of the various heating and / or cooling elements and the temperatures attained by each of the various heating and / or cooling elements. According to this example, the substrate 15 supported by the substrate stage 12 may have a comparatively lower temperature than the periphery of the body, again due to the established temperature gradient.

[0205] In some embodiments, the lower plate 10 may include a device for mixing of fluids and / or reagents disposed on the surface of substrate 15. In some embodiments, the device for mixing of fluids and / or reagents is a contactless mixing device. In some embodiments, the device for contactlessly mixing fluids and / or reagents may be integrated into the body of the lower plate. In other embodiments, the device of contactlessly mixing fluids and / or reagents may in coupled to the body of the lower plate. In some embodiments, the lower plate 10 may include an element or be in communication with an element capable of providing vibrations to the substrate 15 supported by 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 composed of a piezoelectric wafer that generates a mechanical vibration. In some embodiments, a surface transducer is used to distribute or mix a fluid volume on-slide. Suitable devices and methods for contactless mixing are described in PCT Publication No. WO / 2018 / 215844.

[0206] In some embodiments, the lower plate 10 (or the body 16 or housing member 20) is formed by machine or milling a block of solid material. In other embodiments, the lower plate 10 (or the body 16 or housing member 20) is formed through a 3D printing process. In other embodiments, the lower plate 10 or any portion thereof is produced from a mold. The lower plate 10 may be fabricated from any material. For example, the lower plate 10 or any constituent part thereof, may be made from a metal or an alloy; ceramic; glass; glass epoxy laminates, or plastic (e.g. derived from a polymer, a copolymer, or a polymer or co-polymer blend). Examples of suitable polymers include, but are not limited to, polyether ether ketone, polyimides, polyetherimide, polytetrafluoroethylene, polysulfones, polyvinylidene difluoride, and polyphenylene sulfide. Examples of suitable metal materials include aluminum and steel. In some embodiments, if the lower plate is made from a metal, the metal may be coated or uncoated (e.g. coated with a fluoropolymer).

[0207] In some embodiments, the lower plate has a total mass ranging from between about 30 grams to 200 grams. In other embodiments, the lower plate has a total mass ranging from between about 40 grams to 180 grams. In yet other embodiments, the lower plate has a total mass ranging from between about 40 grams to 160 grams. In yet other embodiments, the lower plate has a total mass ranging from between about 40 grams to 140 grams. In other embodiments, the lower plate has a total mass ranging from between about 40 grams to 120 grams. In other embodiments, the lower plate has a total mass ranging from between about 50 grams to 120 grams. In other embodiments, the lower plate has a total mass ranging from between about 50 grams to 100 grams.

[0208] In some embodiments, the lower plate has a thermal capacity ranging from between about 40 J / C to about 110 J / C. In some embodiments, the lower plate has a thermal capacity ranging from between about 40 J / C to about 100 J / C. In some embodiments, the lower plate has a thermal capacity ranging from between about 40 J / C to about 90 J / C. In some embodiments, the lower plate has a thermal capacity ranging from between about 50 J / C to about 80 J / C. In some embodiments, the lower plate has a thermal capacity ranging from between about 50 J / C to about 70 J / C. In some embodiments, the lower plate has a thermal capacity ranging from between about 55 J / C to about 75 J / C.

[0209] With reference to at least FIGS. 7A - 7D, 8A and 8B, in some embodiments the lower plate 10 includes a body 16 and a housing member 20. As noted above, in some embodiments, the body 16 of the lower plate 10 and the housing member 20 are integral with each other and formed from a single monolithic block, e.g. milled or machined from a single monolithic block. In other embodiments, the body 16 of the lower plate 10 is adapted to fit (e.g. releasably fit) within a housing member 20 (see FIGS. 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 into, screwed into, or clamped onto the housing member 20. In other embodiments, the body 16 of the lower plate 10 is removably engageable with the housing member 20.

[0210] For example, the body 16 of the lower plate 10 may be inserted into and removed from the housing member 20 on an as-needed basis. Here, a single type of lower plate 10 may be designed and used with various types of housing members 20, where the various housing members may 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 an upper surface 12C of the substrate stage 12 is recessed relative to an upper surface 20C of the housing member 20 (FIGS. 7A and 8B).

[0211] In other embodiments, the housing member 20 is configured to receive the body 16 such that an upper surface 12C of the substrate stage 12 is level with an upper surface 20C of the housing member 20 (FIG. 7B). In yet other embodiments, the housing member 20 is configured to receive the body 16 such that an upper surface 12C of the substrate stage 12 is raised relative to an upper surface 20C of the housing member 20 (FIG. 7C). In further embodiments, such as depicted in FIG. 7D, the housing member 20 includes one or more heating and / or cooling elements 21 in thermal communication with the body 16, and the one or more heating and / or cooling elements 21 may include any combination of those described herein. Again, with reference 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 provided solely in either one or both of the body 16 of the lower plate 10 and / or the housing member 20.

[0212] In some embodiments, the lower plate 710 has a modular design (see, e.g. FIGS. 26A - 26E). It is believed that a lower plate having a modular design enables the lower plate (or any portion thereof) to be used in a multitude of different specimen processing assemblies 100 and in multiple different ways for multiple different purposes. For instance, a modular lower plate 710 may be adapted to rest on, engage with, or couple to other components of the specimen processing assembly 100.

[0213] In some embodiments, a 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 may rest on, engage with, or couple to the thermal management module 440. FIGS. 26F or example, FIG. 26A - 26G each illustrate a body 716 resting on a thermal management module 440.

[0214] In some embodiments, the body 716 includes a substrate stage 712 that is part of and integral with body 716. In some embodiments, the body 716 may be picked up, transported to, and deposited onto a thermal management module 440 while a substrate is supported by the substrate stage of body 716 (where the body 716 including the substrate stage 712 and the thermal management module 440 together constitute the modular lower plate 710). In some embodiments, the body 716 may be transported between different thermal management modules 440 or between different components of the specimen processing assembly 100 or system 200. In some embodiments, the body 716 may itself include any of the components described herein with regard to the body 16 of the lower plate 10, e.g. one or more heating and / or cooling elements, one or more alignment members, etc. The body 716 may be comprised of any material, including any of those described herein with regard to the lower plate 10.

[0215] In other embodiments, the body 716 includes a lower engagement surface but does not include an integral substrate stage. Rather, the modular lower plate 710 includes a body 716 having a separable substrate stage. In this way, a separable substrate stage may be used as a carrier for a substrate. For example, a substrate may be disposed onto the separable substrate stage and together the substrate and the separable substrate stage pair may be picked up, transported to, and deposited onto a body 716. Following this example further, once an unmasking operation is completed using the modular lower plate 710 (e.g. including the separate substrate stage, the body 716, and a thermal management module), the separable substrate stage (again acting as a carrier for the substrate) may be picked up and transported to other components of the specimen processing assembly 100 or system 200. For example, the separable substrate and separable substrate stage may together be transported to a staining apparatus or a coverslipping apparatus. In some embodiments, a substrate and a separate substrate stage pair remain together throughout all processing steps in system 200.

[0216] In some embodiments, and with reference to FIG. 26A, the modular lower plate 710 includes a body 716 having a substrate stage 712 and a lower engagement surface 711, where the body 716 is in thermal communication with a 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 heating elements and cooling described herein. For example, the thermal management module 440 may include any number of heating and / or cooling elements, e.g. 1 heating element, 1 cooling element, 1 heating element and 1 cooling element, 2 heating elements and 1 cooling element, 2 heating elements, 2 cooling elements, 2 heating elements and 2 cooling elements, etc. In some embodiments, the thermal management module 440 may 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, e.g., FIG. 26B).

[0217] In some embodiments, and with reference to at least FIGS. 26C and 26D, the thermal management module 440 includes one or more electrical contacts and / or one or more ports, e.g. 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. Likewise, the body 716 of the modular lower plate 710 may include one or more complementary electrical contacts for receiving power from the thermal management module 440 and / or one or more complementary ports for receiving fluid from the thermal management module 440.

[0218] For example, and as illustrated in FIG. 26C, the body 716 of the modular lower plate 710 may include one or more bores 401A and 401B into which heating cartridges are inserted. To maintain the ability for the body 716 to be transportable between different processing areas and / or different specimen processing assemblies 100 (or even other system 200 components) and without hindrance from electrical wiring connected to the body 716 or its sub-components, the body 716 may include one or more electrical contacts 442A and 442B such that power may be received from a thermal management module 440. Likewise, and as illustrated in FIGS. 26C and 26D, the body 716 may include one or more fluid channels 411, where fluid may be received into the fluid channels 411 via one or more ports 441A and 441B, the ports 411A and 441B being complementary to the one or more ports on the thermal management module 440. In this way, the body 716 of the modular lower plate 710 may be moved without being directly connected to any fluid delivery lines.

[0219] FIG. 26E illustrates another embodiment where a body 716 is in thermal communication with a thermal management module 440, where the body 716 of the modular lower plate 710 includes bores 401A and 401B into which heating cartridges may be inserted, and where the body 716 of the modular lower plate 710 includes electrical contacts 442A and 442B for providing power to the heating cartridges inserted into bores 401A and 401B. The embodiment depicted in FIG. 26E 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 may be delivered from the thermal management module 440 via electrical contacts 442A and 442B to heating cartridges inserted into bores 401A and 401B to effectuate 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 may provide additional heating to the body 716 of the modular lower plate 710 and / or the substrate stage 712; or may provide cooling to the body 716 of the modular lower plate 710 and / or the substrate stage 712 depending on the temperature of the fluid circulated within fluid channels 411A and 411B.

[0220] FIG. 26F illustrates a substrate 15 positioned on a substrate stage (not shown), the substrate stage being integral with the body 716. In this particular embodiment, the body 716 is shown in thermal communication with a thermal management module 440 including one or more heating and / or cooling elements. FIG. 26F further depicts the body 716 in communication with a transport member 433, such that the transport member 433 is capable of delivering the body 716 to a position above the thermal management module 440 but below an upper plate 30. FIG. 26G illustrates a substrate stage 712 integral with a body 716 of a modular lower plate 710, where the body 716 further includes a lower engagement surface 711, a vacuum port 170, and a vacuum sealing element 171. The body 716 of the modular lower plate 710 is depicted as being supported by and in thermal communication with a thermal management module 440.

[0221] With reference to FIG. 32A, the body 716 may further include one or more attachment members 431A and 431B located on opposite ends (e.g. longitudinal ends) of the body 716. In some embodiments, the one or more attachment members 431A and 431B facilitate the movement of the body 716 by one or more grippers or other devices configured to pick up and / or move the body 716, such as in any one of the x, y, and z coordinate directions. For example, a gripper or other pickup device may be configured to pick up and move a body 716 710 by holding onto the first and second attachment members 431A and 431B. In yet other embodiments, the body 716 may include one or more notches 430A and 430B within the lower surface of body 716 such that a gripper or pickup device (described further herein) may cradle the body 716 from beneath, thus supporting it during any transport operation (see FIG. 32A).

[0222] In some embodiments, one or more sides of the body 716 have a surface which has been roughened or patterned 160 (see FIG. 32B) so as 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 which includes a coating 161 (see FIG. 32C), such as a polymer coating, so as to increase friction between the one or more coated sides the of the body 716 710 and a gripper or other pickup device configured to grasp the body 716.

[0223] As depicted in FIGS. 27A - 27E, a body 716 having a substrate stage 712 and a lower engagement surface 711 may be incorporated within a carrier block 432. The carrier block 432 may have any size or shape. In some embodiments, the carrier block has a size and / or shape which facilitates it being picked up by a gripper device 460 or other pickup device. FIG. 27D illustrates a bottom view of a body 716 incorporated within a carrier block 432. FIG. 27E illustrates a side cutaway view of a body 716 incorporated within a carrier block 432.

[0224] In some embodiments, the body 716 may be fixed within the carrier block 432. In other embodiments, the body 716 is removably engageable with the carrier block 432. In some embodiments, a single body 716 may be used with differently sized and / or shaped carrier blocks 432. In some embodiments, the carrier block 432 is disposable while the body 716 is reusable. In some embodiments, and with reference 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 which emanate from the body 716 (e.g. from the longitudinal ends of the body) and which are integral with the body 716. In some embodiments, the attachment members 431A and 431B on body 716 may have any size and / or shape, provided that the carrier block 432 has a complementary shape which accommodates the attachment members 431A and 431B. In some embodiments, the one or more attachment members 431A and 431B facilitate the alignment of the body 716 within a carrier block 432 and / or secure the body 716 within the carrier block 432 while the combined body 716 and carrier block 432 are moved, such as moved to a thermal management module 440 or to other components of the specimen processing assembly 100 or system 200.

[0225] In some embodiments, the carrier block 432 is comprised of a material selected from polymers, copolymers, metals, etc. In some embodiments, the carrier block 432 is comprised of the same material as that of the body 716. In other embodiments, the body 716 is comprised of a first material (e.g. a metal) while the carrier block 432 is comprised of a second material (e.g. a heat-resistant copolymer). In some embodiments, one or more sides of the carrier block 432 have a surface which has been roughened or patterned so as to increase friction between the one or more sides of the carrier block 432 and the gripper or other pickup device. In other embodiments, one or more sides of the carrier block 432 have a surface which includes a coating, such as a polymer coating, so as to increase friction between the one or more coated sides the of the carrier block 432 and the gripper or other pickup device.

[0226] The movement of a body 716 including carrier block 432 is depicted in FIGS. 27F - 27H. Specifically, FIG. 27F illustrates a body 716 including a carrier block 432 in communication with a carrier pickup member 450. In some embodiments, the carrier pickup member 450 includes a carrier pickup body 452 and a pair of carrier pickup arms 451A and 451B. In some embodiments, the carrier pickup body 452 and carrier pickup arms 451A and 451B are configured to support the edges of carrier block 432. For example, the carrier block 432 may be sized to rest on top of a portion of the carrier pickup body 452 and both carrier pickup arms 451A and 451B. FIG. 27G illustrates a body 716 included a carrier block 432 after the carrier pickup body 450 and carrier block 432 have been moved to a position adjacent a thermal management module 440. As illustrated in FIG. 27G, the carrier block 432 is supported by at least carrier pickup arms 451A and 451B. FIG. 27H illustrates a body 716 including a carrier block 432 after the carrier 432 has been moved via a pick-up device (described herein) and positioned on top of a thermal management module 440. As illustrated in FIG. 27H, the carrier pickup arms 451A and 451B are no longer in communication with, e.g. supporting, the edges of carrier block 432. As depicted, the carrier pickup device and carrier pickup member may be moved away from the thermal management unit 440, while leaving the carrier block 432 and body 716 supported by the thermal management module 440.

[0227] Although not depicted, in some embodiments, the lower plate may include two or more substrate stages. In other embodiments, the lower plate may include three or more substrate stages. In yet other embodiments, the lower plate may include four or more substrate stages. In further embodiments, the lower plate may include 10 or more substrate stages. By way of example, FIG. 22A illustrates a lower plate having two substrate stages 12 arranged parallel to each other. In embodiments where the lower plate includes two or more substrate stages, in some embodiments, an upper plate will include features which are complementary to the features of the lower plate having the two or more substrate stages (see, for example, FIG. 22B which includes two cavities 32 adapted to receive at least the substrates 15 of FIG. 22A). In such embodiments, complementary upper and lower plates may be brought together to form a chamber, whereby the chamber is adapted to process two or more substrates together. In some embodiments, the upper plate and the lower plate may be configured such that there is a divider between each substrate stage. In this way, the divider may act to provide separate processing chambers formed from a single upper plate and a single lower plate. In some embodiments, each of the multiple substrates may be heated by the same heating element or each may be heated independently with a separate heating element, such as separate heating elements embedded within each substrate stage. Other features of the chamber and how the chamber is formed from lower and upper plates having certain complementary features are described herein.UPPER PLATE

[0228] The specimen processing assemblies of the present disclosure include at least one upper plate, such as those illustrated in FIGS. 9A - 9H. With reference to FIG. 9A, in some embodiments, the upper plate 30 includes a body 33 having an upper engagement surface 31. In some embodiments, the upper plate 30 further includes one or more cavities 32 (at least FIG. 9A), for example one or more cavities recessed into the body 33. In some embodiments, the upper plate may include a body 33 having an upper engagement surface 31, but unlike the embodiment depicted in FIG. 9A, the upper plate 30 may not include a recessed cavity (see, for example, FIG. 10E).

[0229] In some embodiments, the upper plate 30 includes elements which are complementary to features of the lower plate 10. In some embodiments, the features of the upper plate 30 at least partially conform to features present on the lower plate 10. In particular, the upper plate 30 includes elements which are adapted to conform to those features of the lower plate 10 such that when the upper and lower plates are brought together, a chamber may be formed. In this regard, the upper and lower plates may be considered the complements of one another, such that the upper plate is adapted to accommodate features of the lower plate (or vice versa).

[0230] As with the lower plate 10, the upper plate 30 may have any size or shape, provided the upper plate 30 is complementary to the lower plate 10. For example, as depicted in FIGS. 9A - 9H, the upper plate 30 may have a generally rectangular shape whereby both the rectangular upper and lower plates are complementary to each other. Alternatively, and as illustrated in FIGS. 6A and 6B, the upper and lower plates 30 and 10, respectively, may both have a wedge-based shape, whereby the upper plate 30 includes an upper engagement surface 31 having a size and / or shape which is complementary to the size and / or shape of the lower engagement surface 11 or 711, but where the upper and lower surfaces have differing conformations.

[0231] The cavity 32 itself may have any size or shape. In some embodiments, the cavity 32 has a size and / or shape adapted to accommodate features of the lower plate 10, e.g. a substrate stage 12 and any substrate 15 disposed on the substrate stage 12. In this regard, the cavity 32 may have a shape and size which is complementary to those portions of the lower plate 10 which are raised relative to a lower engagement surface 11 or 711. In some embodiments, the portions of the lower plate 10 which are raised relative to a lower engagement surface 11 may have a first conformation, while the cavity 32 may be adapted to have a second conformation which is the opposite of the first conformation (and optionally including additional headspace 40 to accommodate any substrate, fluids, and / or reagents disposed on the substrate). Additionally, in some embodiments, the cavity 32 may be adapted to accommodate any fluids, reagents, or specimens disposed on a surface of substrate 15 and / or to further accommodate headspace, for example a predetermined dead volume above a substrate surface 15C.

[0232] FIGS. 9C and 9D provide cut-away views of a body 33, illustrating non-limiting examples of a cavity 32. In some embodiments, the cavity 32 is about the same size as or larger than a substrate 15. In some embodiments, the cavity 32 has a volume that is at least the same as a volume of a substrate (e.g. a volume that is the same as a volume of the substrate and a predefined amount of headspace volume). In some embodiments, the cavity 32 is sized to have a volume which would accommodate not only a substrate, but at least a portion of a substrate stage 12. By way of a non-limiting example, FIG. 9C depicts a cavity 32 having a volume larger than a volume of a substrate 15. For instance, the additional volume available may accommodate at least a portion of a substrate stage 12 upon which the substrate 15 is supported. By way of another non-limiting example, FIG. 9D illustrates a cavity 32 sized to at least accommodate a substrate (e.g. a microscope slide) and any materials disposed on the surface of the substrate, including fluids, reagents, and / or samples. The cavity also includes a headspace 40 having a predefined volume. While FIGS. 9C - 9F depict a cavity 32 having a monolithic recess, in some embodiments, the cavity 32 may be tiered, such as depicted in FIGS. 10A - 10D. In some embodiments, a tiered cavity 32 may conform to the features of the lower plate 10, including those features raised relative to a lower engagement surface 11.

[0233] With reference to FIG. 9B, in some embodiments, the upper plate 30 includes a sealing member 39 which is raised relative to the engagement surface 31, for example protruding from the upper engagement surface 31. In some embodiments, the sealing member 39 may be comprised of 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 monolithic block or 3D printed as a single unit. In some embodiments, the sealing member 39 is adapted to fit within and / or engage the walls of a groove 13 in a lower plate 10. Although not depicted, in other embodiments, the upper plate may alternatively include a groove similar to groove 13 within the lower plate 10 to accommodate a seal body, for example the upper plate may include a groove having walls capable of engaging a seal body (such as those described herein).

[0234] In some embodiments, the upper plate 30 may include one or more heating elements and / or one or more cooling elements, for example one or more elements adapted to heat and / or cool the material of the upper plate. In some embodiments, the one or more heating and / or cooling elements may be the same or different as those described for use in relation to the lower plate (see description above). In some embodiments, an active cooling element includes a plurality of fluid flow channels to facilitate the flow of a coolant and thus provide active cooling of the sample, the upper plate, and / or the lower plate. In some embodiments, one or more heating and / or cooling elements are integrated or embedded within the upper plate 30 (see, e.g., FIGS. 9G and 9H). In other embodiments, one or more heating and / or cooling elements are mounted external to one or more surfaces of the body 33 of the upper plate 30. In some embodiments, the upper plate 30 may include an integral heat sink, e.g. one formed into the side walls of the body 33. Additional embodiments illustrating the use of heating elements and / or cooling elements in the upper and lower plates are illustrate din FIGS. 21A - 21D.

[0235] In some embodiments, the upper plate 30 may include one or more heating and / or cooling elements 21 (see FIGS. 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, FIGS. 29A and 29C each depict an upper plate 30 having a body 33, whereby 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. Likewise, the body 33 of the upper plate 30 may include any combination of heating and / or cooling elements, e.g. 1 heating and / or cooling element; 1 heating 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.

[0236] In some embodiments, the one or more heating and / or cooling elements 21 may have any arrangement within the body 33 of the upper plate 30. For illustrative purposes only, FIG. 29B illustrates one or more heating elements 21A arranged parallel to one or more cooling elements 21B. In some embodiments, the one or more heating and / or cooling elements may 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 versus elements 411A and 411B as in FIG. 29F), may be spaced evenly or randomly, etc. In some embodiments, one or more heating and / or cooling elements may be positioned opposite the cavity 32, while additional heating and / or cooling elements are positioned beside the one or more heating and / or cooling elements positioned opposite the cavity 32, whereby the additional heating and / or cooling elements flank the one or more heating and / or cooling elements positioned opposite the cavity (see FIG. 29D).

[0237] The body 33 of upper plate 30 may include one or more bores into which a heating module, e.g. a heating cartridge may be inserted. In some embodiments, any number of bores may be provided within the body 33 of an upper plate 30, e.g. 1 bore, 2 bores, 3 bores, 4 bores, 6 bores, 8 bores, 12 bores, etc. The bores may be arranged in any manner within the body 16 of the lower plate. As such, the body 16 may accommodate 1, 2, 3, 4, 6, 8, 12, etc. heating cartridges. For example, FIGS. 29D and 29E each illustrates a body 33 having two bores 401A and 401B into which heating cartridges may be inserted.

[0238] In some embodiments, one or more bores may be evenly spaced apart from one another. In other embodiments, the one or more bores may be randomly spaced. In some embodiments, the bores are open to the longitudinal side of the upper plate 30. Alternatively, in in other embodiments, the bores may be open on the "short side" of the lower plate, as illustrated in FIG. 29E. The bores themselves may independently have any size and / or diameter.

[0239] In some embodiments, each of the one or more heating cartridges inserted into the body 33 may be independently operable. For example, assuming that a body 33 of an upper plate 30 includes three heating cartridges inserted into three bores, each of the three heating cartridges may be independently operable, for example heating cartridges 1 and 3 may be operated at 80% of their power rating while cartridge 2 is operated at 40% of its power rating.

[0240] In other embodiments, the one or more heating elements and / or cooling elements 21 embedded within the body 33 of the upper plate 30 include one or more fluid channels, where the one or more fluid channels serve to circulate a fluid within the body 33 of the upper plate 30. In some embodiments, the fluid is a 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 may have any configuration and may be independently operable. For example, and with reference to FIGS. 29D and 29F, the one or more fluid channels 411A and 411B may be arranged as two independent parallel conduits which independently permit the flow of a suitable fluid through the body 33. In this particular example, the fluid channels 411A and 411B may each be configured for cooling, may each be configured for heating, or one may be configured for heating while the other is configured for cooling. Likewise, the fluid channels 411A and 411B, while both configured for heating, may be configured to heat at different temperatures.

[0241] Alternatively, the one or more fluids channels may include a network 412 of fluid channels, such as those depicted in FIG. 29G. The network 412 of fluid channels may include one or more independently controllable inlets 414, outlets 413, or valves (not depicted) so as to direct the circulation along a particular fluid flow path, or to provider circulation within only a portion of the network of channels. In some embodiments, the network of fluid channels may be in communication with a fluid reservoir (not depicted), and where the fluid reservoir may be heated or cooled as needed.

[0242] In some embodiments, any of the heating and / or cooling elements within the upper plate 30 may be operated in conjunction with those heating and / or cooling elements within the lower plate 10 or with any thermal management module. As a result, the skilled artisan will appreciate that by controlling the various heating and / or cooling elements present in the upper and lower plates and / or in the thermal management module, that the temperature of the substrate stage, any substrate supported by the substrate stage, and / or the chamber may be regulated. For example, it is possible to operate the various heating and / or cooling elements present in the upper and lower plates such that the substrate, or the specimen disposed on the substrate, remains the coldest structure within the chamber, for example the substrate and / or the specimen disposed on the substrate are maintained at a temperature lower than the upper plate, the lower plate, ports, and / or any other structure within the chamber formed from the upper and lower plates. In some embodiments, and with reference to FIG. 40C, a temperature of the cavity 32 of the upper plate 30 is maintained at a temperature F which is the same as the temperature E of any of the surrounding portions of body 33 of the upper plate 30. In other embodiments, and with reference to FIG. 40C, a temperature of the cavity 32 of the upper plate 30 is maintained at a temperature F which is lower than the temperature E of any of the surrounding portions of body 33 of the upper plate 30. In other embodiments, and with reference to FIG. 40C, a temperature of the cavity 32 of the upper plate 30 is maintained at a temperature F which is higher than the temperature E of any of the surrounding portions of body 33 of the upper plate 30.

[0243] In some embodiments, the upper plate may include one or more ports. In some embodiments, the ports comprise a seal or a pressure valve such that gases and / or steam may be released or introduced (such as from a chamber formed from complementary upper and lower plates). In some embodiments, the one or more ports are configured such that gases and / or steam may be introduced into a chamber formed from the upper and lower plates. In some embodiments, the ports are connected to a gas and / or steam source and the valves may be independently operated such that steam may be introduced, such as into a chamber formed between the upper and lower plates (see FIG. 21D). In some embodiments, gas and / or steam is introduced to at least partially heat the substrate and / or sample. In other embodiments, gas and / or steam is introduced to pre-pressurize a chamber formed by the upper and lower plates. In other embodiments, the one or more ports are configured for the introduction of one or more fluids and / or reagents.

[0244] 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, e.g. an external force applied by one or more force generating members. In some embodiments, the one or more force generating members include motors, springs, screws, levers, pistons (e.g. a mechanically, an electrically, pneumatically, or hydraulically actuated pistons), cam mechanisms, or any combination thereof. In some embodiments, the externally applied force ranges from between about 5 Newtons to about 3000 Newtons. In other embodiments, the externally applied force ranges from between about 10 Newtons to about 2000 Newtons. In other embodiments, the externally applied force ranges from between about 10 Newtons to about 1000 Newtons. In other embodiments, the externally applied force ranges from between about 10 Newtons to about 500 Newtons. In yet other embodiments, the externally applied force ranges from between about 10 Newtons to about 250 Newtons. In further embodiments, the externally applied force ranges from between about 20 Newtons to about 150 Newtons. In some embodiments, the force generating member exerts 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 either less than a predetermined threshold pressure or an amount which does not exceed any predetermined threshold pressure of the internal environment within a chamber formed from the complementary upper and lower plates. In this regard, the force exerted by the force generating member is less than the forces pushing the upper and lower plates apart and, as a result, the force generating member may give way or slip such that pressure may be relieved from within the formed chamber.

[0245] 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, e.g. to evenly distribute a received force evenly over the upper plate 30. In some embodiments, and as illustrated in FIG. 9E, the at least one pressure member includes two parallel bars 34A and 34B. In some embodiments, the parallel bars 34A and 34B run along the longitudinal sides 41 of the body 33 of the upper plate 30 (see also FIGS. 11A and 11B). In other embodiments, and with reference to FIG. 9F, the at least one pressure member includes a pressure plate 35, e.g. a monolithic pressure plate coupled to an upper surface 33A of the upper plate 30. In some embodiments, the pressure plate 35 includes a surface area which is larger than a surface area of the upper surface 33A of the upper plate 30. In some embodiments, the pressure plate 35 includes a surface area which is smaller than a surface area of the upper surface 33A of the upper plate 30.

[0246] Although not depicted, in some embodiments, an upper surface 33A of the body 33 (or, for that matter, the pressure plate 35) may include one or more mounting points adapted to couple the upper plate to a support member or a sub-assembly.

[0247] In some embodiments, and as depicted in FIGS. 9G and 9H, the pressure plate 35 is adapted to fit within a void of body 33. In some embodiments, the pressure plate 35 which is adapted to fit within a void of the body 33 frictionally engages a raised portion of the body 33 of the upper plate 30. In other embodiments, the pressure plate 35 which is adapted to fit within a void of the body 33 is fixedly secured to the body 33 of the upper plate 30, e.g. glued into, screwed into, or clamped onto 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 further an integral seal 38.

[0248] In some embodiments, the upper plate 30 is formed by machine or milling a block of solid material. In other embodiments, upper plate 30 is formed through a 3D printing process. In other embodiments, the upper plate 30 or any portion thereof is produced from a mold. The upper plate 30 may be fabricated from any material. For example, the upper plate 30 or any constituent part thereof, may be made from a metal or an alloy; ceramic; glass; or plastic (e.g. derived from a polymer, a copolymer, or a polymer or co-polymer blend). Examples of suitable polymers include, but are not limited to, polyether ether ketone, polyimides, polyetherimide, polytetrafluoroethylene, polysulfones, polyvinylidene difluoride, and polyphenylene sulfide. Examples of suitable metal materials include aluminum and steel. In some embodiments, if the upper plate is made from a metal, the metal may be coated or uncoated (e.g. coated with a fluoropolymer).

[0249] In some embodiments, the upper plate has a total mass ranging from between about 30 grams to 200 grams. In other embodiments, the upper plate has a total mass ranging from between about 40 grams to 180 grams. In yet other embodiments, the upper plate has a total mass ranging from between about 40 grams to 160 grams. In yet other embodiments, the upper plate has a total mass ranging from between about 40 grams to 140 grams. In other embodiments, the upper plate has a total mass ranging from between about 40 grams to 120 grams. In other embodiments, the upper plate has a total mass ranging from between about 50 grams to 120 grams. In other embodiments, the upper plate has a total mass ranging from between about 50 grams to 100 grams.

[0250] In some embodiments, the upper plate has a thermal capacity ranging from between about 40 J / C to about 110 J / C. In some embodiments, the upper plate has a thermal capacity ranging from between about 40 J / C to about 100 J / C. In some embodiments, the upper plate has a thermal capacity ranging from between about 40 J / C to about 90 J / C. In some embodiments, the upper plate has a thermal capacity ranging from between about 50 J / C to about 80 J / C. In some embodiments, the upper plate has a thermal capacity ranging from between about 50 J / C to about 70 J / C. In some embodiments, the upper plate has a thermal capacity ranging from between about 55 J / C to about 75 J / C.ADDITIONAL SPECIMEN PROCESSING ASSEMBLY COMPONENTS

[0251] In some embodiments, the specimen processing assemblies of the present disclosure include (i) a lower plate; (ii) an upper plate which is 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 sub-assembly, a support member, a force generating member, a substrate loader, etc.

[0252] In some embodiments, the specimen processing assemblies include one or more sub-assemblies, e.g. rails, to which one of the lower and / or upper plates may be independently coupled (either directly or indirectly). In some embodiments, the one or more sub-assemblies enable the independent movement of the lower and / or upper plates in any of the x, y, and z-coordinate directions. For example, and with reference to at least FIGS. 12A and 12B, the specimen processing assemblies of the present disclosure may, in some embodiments, include a lower plate 10 movably coupled to a sub-assembly 101. In some embodiments, the sub-assembly is a rail. In some embodiments, the sub-assembly includes a motor coupled to a belt or a screw, where the belt or screw may be directly or indirectly coupled to the lower plate 10.

[0253] In some embodiments, the sub-assembly 101 may be linear or curvilinear, e.g. arcuate, semi-circular, etc. In other embodiments, the sub-assembly 101 may have a complex shape including one or more portions which are linear and one or more portions which are curvilinear, e.g. two portions that are linear with an intermediary portion that is arcuate. In some embodiments, two or more lower plates 10 may be coupled to a single sub-assembly 101, where each of the two or more plates 10 may move independently or may be linked such that they move in tandem.

[0254] In some embodiments, the lower plate 10 may be directly coupled to the sub-assembly 101, such as within a track along the top and / or sides of the sub-assembly 101 (see FIGS. 12A and 12B). In this way, the lower plate 10 may directly slide or be moved within the track along the sub-assembly 101, such as along the plane of the sub-assembly 101.

[0255] In other embodiments, the lower plate 10 may be indirectly coupled to the sub-assembly 101. For example, a support member 115A may be coupled to (i) a portion of the lower plate 10, and (ii) a track of the sub-assembly 101. In this particular embodiment, the support member 115A facilitates the movement of the lower plate 10 along the sub-assembly 101. In some embodiments, the support member 115A is configured such that a plane defined by the upper surface of substrate 15 remains horizontal, for example parallel to the ground, regardless of the movement of the lower plate 10 along the sub-assembly 101. In some embodiments, the support member 115A includes one or more springs.

[0256] In some embodiments, the sub-assembly 101 may be positioned horizontally. This is illustrated in FIG. 12A, where the sub-assembly 101 is positioned horizontally within the x,y plane. In this embodiment, the horizontal movement of the lower plate 10 along the length of the sub-assembly 101 would be within the x,y plane. FIG. 12A also depicts a substrate stage 12 having an upper surface which is parallel to the ground. By virtue of the rail being positioned parallel to the ground, any substrate 15 supported by the substrate stage 12, and any specimens, fluids and / or reagents disposed thereon, remains parallel to the ground (for example within the x,y plane of FIG. 12A) throughout the movement of the lower plate 10.

[0257] In other embodiments, one end of the sub-assembly 101 may be raised along the z-axis relative to another end. For example, FIG. 12B illustrates a sub-assembly 101 having a first end 113 and a second end 114, whereby the first end 113 is raised along the z-axis relative to the second end 114 (hereinafter referred to as being "offset from the horizontal" or "horizontally offset"). In such embodiments, a component of the movement of the lower plate 10 along a horizontally offset sub-assembly 101 will be along the z-axis, for example there is a vertical component to the movement. This is further illustrated, for example, in FIGS. 16A and 16B, which illustrate that a lower plate 10 may be moved along a sub-assembly 101, such as from end 114 to end 113, and from a first position, to a second position, and any intermediary position therebetween.

[0258] In some embodiments, the horizontal offset may be at an angle ranging from between about 5 degrees to about 70 degrees. In other embodiments, the horizontal offset angle may range from between about 5 degrees to about 60 degrees. In yet other embodiments, the horizontal offset angle may range from between about 5 degrees to about 50 degrees. In yet other embodiments, the horizontal offset angle may range from between about 10 degrees to about 50 degrees. In yet other embodiments, the horizontal offset angle may range from between about 15 degrees to about 50 degrees. In further embodiments, the horizontal offset angle may range from between about 20 degrees to about 50 degrees. In yet further embodiments, the horizontal offset angle may range from between about 20 degrees to about 45 degrees.

[0259] In embodiments where the sub-assembly 101 is offset from the horizontal, a support member 115A may be adapted to orient the lower plate 10, and hence the upper surface of the substrate stage 12, such that any substrate positioned thereon remains parallel to the ground and within the x,y plane. In other embodiments, the lower plate itself is configured such that either a lower engagement surface remains parallel to the ground or a surface of a substrate stage remains parallel to the ground.

[0260] In some embodiments, the upper plate 30 may be coupled to a sub-assembly. In some embodiments, and with reference to FIG. 14A, the upper plate 30 may be coupled to a sub-assembly such that it is fixed in space, such as through a support member 115B. In those embodiments where the lower plate 10 is fixed, the upper plate may be movably coupled to a sub-assembly (movable in any of the x, y, and z directions). In these embodiments, the upper plate 30 may be coupled to the sub-assembly in a manner that allows its movement along any of the x, y, and z-axes. Alternatively, the upper plate 30 may be coupled in a manner that allows its movement only within a z-axis.

[0261] In some embodiments, the upper plate may be coupled directly or indirectly to one or more force generating members. In some embodiments, the one or more force generating members include motors, springs, screws, levers, pistons (e.g. a mechanically, pneumatically, electrically, or hydraulically actuated pistons), cam mechanisms, or any combination thereof. In some embodiments, the force generating member facilitates either movement of the upper plate along the z-axis (e.g. to move the upper plate toward a lower plate) or the exertion of a force along the z-axis (such that a force is exerted to maintain the upper and lower plates in contact with one another, even when a chamber formed from the upper and lower plates is pressurized). In some embodiments, the one or more force generating members exert a predetermined force onto the upper plate. In some embodiments, this predetermined force is limited such that should pressure within the chamber exceed a predetermined threshold pressure, the force exerted by the force generating member is overcome and the force generating member will slip or give way to relieve pressures above the predetermined threshold pressure that might develop within the chamber. For example, where the one or more force generating members includes a spring or a piston, the spring or piston may be configured to exert a predetermined amount of force which is less than any force exerted when the chamber exceeds a 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 such that such that a predetermined threshold pressure with the chamber is not exceeded.

[0262] In some embodiments, the specimen processing assemblies may include a substrate loader 302 (see, e.g., FIG. 19A) such that substrates may be moved to or removed from a lower plate 10. Additional aspects of substrate loaders and examples of their utilization in certain specimen processing assemblies is described further herein.

[0263] In some embodiments, the specimen processing assemblies include one or more substrate and / or lower plate leveling devices. The substrate and / or lower plate leveling devices may include, for example, one or more adjustable feet or other adjustment protrusions or springs such that a substrate may be maintained in a substantially horizontal position during processing, as described herein.CHAMBERS FORMED FROM THE UPPER AND LOWER PLATES

[0264] As noted above, the specimen processing assemblies of the present disclosure may include one or more chambers. In some embodiments, each chamber is formed from a lower plate and an upper plate, where the upper plate is complementary to the lower plate. In some embodiments, the chamber formed from the complementary upper and lower plates 30 and 10, respectively, is adapted to at least enclose an upper surface 15A of substrate 15 and any fluids, reagents, and / or samples disposed thereon (see, for example, FIGS. 6B and 10A - 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 provided 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 provided within the formed chamber.

[0265] As set forth herein, the upper plate 30 and the lower plate 10 are each configured to have sizes and / or shapes which are complementary to one another such that features present on one of the lower or upper plates are accommodated by the other of the upper or lower plate. In some embodiments, a chamber may be formed when the complementary upper and lower engagement surfaces 31 and 11 (or 711) of the upper and lower plates 30 and 10 (or 710), respectively, are brought into contact with one another. In some embodiments, the contact between the upper and lower engagement 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, the formation of the seal is further facilitated through the application of an external force to one or both of the upper and lower plates, as described further herein.

[0266] In some embodiments, the chamber formed by the lower and upper plates 10 and 30, respectively, provides for an environment which may be controlled according to user preferences. For example, the chamber may be configured such that an environment within or surrounding the formed chamber is different than an environment external to the chamber, for example an environment outside of the lower and upper plates. In some embodiments, the formed chamber allows specimens, fluids, and / or reagents disposed on the surface of a substrate to be treated at elevated temperatures (for example temperatures greater than room temperature) and / or at elevated pressures (for example pressures greater than atmospheric), as described further herein.

[0267] In some embodiments, the chamber formed from the complementary upper and lower plates may be internally pressurized, such as by heating a fluid present within the chamber and / or introducing gas and / or steam into the chamber, such as through one or more ports in communication with the formed chamber. For example, a fluid present on a substrate or within a reservoir within the chamber may be heated to increase the pressure within 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 may contact the fluid reservoir (regardless of its location) and heat the fluid within the reservoir to generate steam. The generated steam may then be used to heat and / or pressurize the chamber.

[0268] Alternatively, a gas and / or steam may be introduced into the chamber (such as through one or more ports in communication 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 modulated by turning off and on one or more heating elements and / or cooling elements, introducing additional gas and / or steam into the chamber through one or more ports, releasing gas and / or steam through one or more ports, or any combination thereof. In some embodiments, the specimen processing device and / or the chamber includes one or more safety mechanisms for preventing over-pressurization, e.g. vent ports, a force generating member which may exert a force less than the forces exerted outward from the internal pressurization of the chamber.

[0269] In some embodiments, the chamber is adapted to sustain temperatures ranging from between about 70°C to about 220°C. In other embodiments, the chamber is adapted to sustain temperatures ranging from between about 90°C to about 200°C. In yet other embodiments, the chamber is adapted to sustain temperatures ranging from between about 95°C to about 180°C. In further embodiments, the chamber is adapted to sustain temperatures ranging from between about 100°C to about 170°C. In even further embodiments, the chamber is adapted to sustain temperatures ranging from between about 110°C to about 160°C. In yet further embodiments, the chamber is adapted to sustain temperatures ranging from between about 120°C to about 150°C.

[0270] In some embodiments, a pressure ranging from between about 95 kPa to about 2000 kPa is maintained within the chamber during heating. In other embodiments, a pressure ranging from between about 100 kPa to about 1600 kPa is maintained within the chamber during heating. In yet other embodiments, a pressure ranging from between about 150 kPa to about 1050 kPa is maintained within the chamber during heating. In further embodiments, a pressure ranging from between about 190 kPa to about 850 kPa is maintained within the chamber during heating. In even further embodiments, a pressure ranging from between about 260 kPa to about 750 kPa is maintained within the chamber during heating. In yet further embodiments, a pressure ranging from between about 300 kPa to about 700 kPa is maintained within the chamber during heating.

[0271] In some embodiments, a force generating member is utilized to exert a force onto the chamber, such as a force generating member which exerts a force onto the upper plate through one or more pressure members in communication with the upper plate. In this manner, the force exerted by the force generating member allows for the pressurized internal environment of the chamber to be maintained. As also noted above, the force exerted by the force generating member may be predetermined. In some embodiments, the predetermined force exerted by any force generating member is less than any predetermined threshold pressure. Alternatively, the predetermined force exerted by the force generating member is programmed not to exceed any predetermined threshold pressure. In this regard, should the pressure within the chamber exceed the predetermined threshold pressure, and given that the predetermined force is set to be lower than any predetermined threshold pressure or set not to exceed the predetermined threshold pressure, the force generating member will give way such that the chamber may release or vent excess pressure, for example the upper and lower plates may at least partially separate from each other so as to permit gases and / or steam to be released to an external environment.

[0272] By way of example, if the chamber or substrate, fluids, and / or reagents disposed within the chamber are heated to about 160°C and the predetermined threshold pressure is set as 750 kPa, then a force generating member is configured to exert an amount of force which is either less than the predetermined threshold pressure or is configured to exert an amount of force which does not exceed the predetermined threshold pressure. Following this example, should the pressure exceed the predetermined threshold pressure of 750 kPa (e.g. the chamber reaches a pressure of 760 kPa), then the force generating member will give way to permit release of the excess gas and / or steam. As noted herein, the upper and / or lower plates may further include one or more ports and / or valves to permit the release of gas and / or steam from the chamber should the predetermined threshold pressure be exceeded.

[0273] In some embodiments, the force generating member is configured to give way if the pressure within the chamber exceeds 1250 kPa. In other embodiments, the force generating member is configured to give way if the pressure within the chamber exceeds 1050 kPa. In yet other embodiments, the force generating member is configured to give way if the pressure within the chamber exceeds 950 kPa. In further embodiments, the force generating member is configured to give way if the pressure within the chamber exceeds 900 kPa. In further embodiments, the force generating member is configured to give way if the pressure within the chamber exceeds 850 kPa. In further embodiments, the force generating member is configured to give way if the pressure within the chamber exceeds 800 kPa. In even further embodiments, the force generating member is configured to give way if the pressure within the chamber exceeds 750 kPa. In yet even further embodiments, the force generating member is configured to give way if the pressure within the chamber exceeds 700 kPa. In yet even further embodiments, the force generating member is configured to give way if the pressure within the chamber exceeds 650 kPa. In yet even further embodiments, the force generating member is configured to give way if the pressure within the chamber exceeds 600 kPa. In yet even further embodiments, the force generating member is configured to give way if the pressure within the chamber exceeds 500 kPa. In still other embodiments, the force generating member is configured to give way if the pressure exceeds 200 kPa, 300 kPa or 400 kPa. The predetermined threshold pressure at which the force generating member gives way can be set to any value that prevents chamber pressures from exceeding values that are deemed unsafe. For example, the predetermined threshold pressure may vary depending upon the materials from which the chamber is constructed, local safety regulations, and combinations thereof.

[0274] In some embodiments, one of the lower and / or upper plates 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, one or more temperature sensors are placed below the substrate. In other embodiments, the one or more temperature sensors are placed on the substrate, such as at one end of the substrate or on an edge of the substrate. In some embodiments, the one or more temperature sensors are placed on the sample disposed on the substrate.

[0275] The complementarity of the shapes and / or sizes of features of the upper and lower plates and how this complementary facilitates the formation of a chamber therebetween is further illustrated in FIGS. 10A - 10C. For example, FIG. 10A illustrates upper and lower plates 30 and 10, respectively, that have been brought together and which are contacting one another. In some embodiments, the upper and lower plates 30 and 10, respectively, contact each other at the interface of the lower engagement surface 11 and the upper engagement surface 31. As further depicted in FIG. 10A, the recessed cavity 32 within the upper plate 30 is sized to accommodate at least substrate 15 and / or a portion substrate stage 12, as well as any specimens, fluids, and / or reagents, disposed on the upper surface of the substrate 15. In some embodiments, the recessed cavity 32 further includes a predetermined amount of headspace, e.g. space surrounding the substrate 15. In some embodiments, the chamber has a volume ranging from between about 14cm 3< to about 25cm 3< . In some embodiments, the chamber has a volume ranging from between about 15cm 3< to about 24cm 3< . In some embodiments, the chamber has a volume ranging from between about 16cm 3< to about 23cm 3< . In some embodiments, the chamber has a volume ranging from between about 17cm 3< to about 22cm 3< . In some embodiments, the chamber has a volume ranging from between about 18cm 3< to about 21cm 3< . In some embodiments, the chamber has a volume ranging from between about 19cm 3< to about 20cm 3< .

[0276] Likewise, FIG. 10B illustrates upper and lower plates 30 and 10, respectively, that have been brought together and which are contacting one another, where the upper engagement surface 31 contacts the lower engagement plate 11 and / or any seal body disposed within groove 13 of the lower plate 10 (seal body not depicted). Additionally, FIG. 10B shows a recessed cavity 32 within the body 33 of the upper plate 30, where the recessed cavity 32 is again sized to accommodate at least the substrate 15 and / or a portion of the substrate stage 12. In some embodiments, the recessed cavity 32 of FIG. 10B includes additional headspace 40 surrounding the substrate 15 as compared with the recessed cavity 32 of FIG. 10A.

[0277] FIG. 10C further illustrates that the body 16 of the lower plate 10 may be in communication with a heating element 21, such as any of those types of heating elements described herein. The embodiments depicted in FIGS. 10A - 10C may further include one or more pressure members in communication with the upper plate 30 (such as to receive an externally applied force and to confer 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 a lower plate 10 so as to passively dissipate heat; cooling channels in thermal communication with one of the upper and / or lower plates so as to actively remove heat), and / or one or more heating elements (e.g. one or more heating elements embedded within or in communication with the upper plate 30 and / or the lower plate 10 so as to heat the upper and lower plates or any materials disposed on the surface of the substrate when positioned within the formed chamber) (see, e.g., FIG. 10D).

[0278] In some embodiments, and with reference to FIG. 10E, a chamber may be formed from complementary upper and lower plates 30 and 10, respectively, where the lower plate 10 includes a lower engagement surface 11 and a substrate stage 12 recessed relative to the lower engagement surface 11, and where the upper plate 30 includes an upper engagement surface 31 but no recessed cavity. FIG. 10F illustrates an alternative embodiment where the upper plate 30 includes a cavity recessed relative to an upper engagement surface 31 and a lower plate 10 including a substrate stage 12 recessed relative to the lower engagement surface 11. When the two plates are brought into contact with one another, a chamber is formed 32.

[0279] FIG. 11A illustrates a non-limiting embodiment illustrating the positioning of a lower plate 10 and an upper plate 30 relative to each other such that complementary features of each of the lower and upper plates may align. In some embodiments, a lower plate 10 is positioned such that a least a portion of the lower engagement surface 11 of the lower plate 10 aligns with a portion of the upper engagement surface 31 of the upper plate 30. FIG. 11B depicts a lower plate 10 and an upper plate 30 in physical communication with one another, for example at least a portion of the lower engagement surface 11 of the lower plate 10 is in contact with a portion of the upper engagement surface 31 of the upper plate 30, thereby forming a chamber enclosing at least a portion of the substrate 15 and any specimens, fluids, and / or reagents, disposed thereon.

[0280] In some embodiments, the chamber formed from the upper and lower plates 30 and 10, respectively, is in communication with one or more heating elements and / or cooling elements such that any substrate or sample positioned with the chamber may be heated and / or cooled; or such that any fluid present within the chamber (such as on the substrate or in a separate reservoir) may be heated to increase the pressure within the chamber. In some embodiments, at least one of the upper plate 30 or the lower plate 10 includes a heating element. In some embodiments, at least one of the upper plate 30 or the lower plate 10 comprise 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 which utilizes 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 heatsink coupled to a 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, a cooling element and a heating element are operated together so as to provide uniform or homogenous temperature throughout the substrate and / or the sample (e.g. each may be turned off or on as needed and in a controlled manner so as to provide a substrate or a sample having a homogeneous temperature distribution). In some embodiments, it is believed that a homogeneous temperature distribution in the sample may be attained when using a heating surface below the substrate, or by introducing an additional heating source above the sample (see FIGS. 21A and 21B). In some embodiments, the chamber includes one or more pressure and / or temperature sensors.

[0281] FIGS. 30A to 30D each illustrate alternative embodiments of a chamber formed from upper and lower plates 30 and 10, respectively, and further illustrate the relative positioning of one or more heating and / or cooling elements 21 within the upper and lower plates and the relative positioning of the heating and / or cooling elements to the chamber and / or to a substrate disposed within the chamber. For example, FIG. 30A illustrates that an upper plate 30 may include one or more heating and / or cooling elements 21 embedded within a body 33 of the upper plate; while one or more heating and / or cooling elements 21 are also embedded within a body 16 of the lower plate.

[0282] FIG. 30B illustrates an embodiment where the upper and lower plates each include fluid flow channels (411A through 411), where the fluid flow channels are arranged beneath recess 32 and beneath the substrate stage 12. The fluid flow channels 411A - 411D may each be independently operated. For instance, the type of fluid, the fluid flow rate, the temperature of the fluid, etc. may each independently controlled for each of the fluid flow channels 411A through 411D. Heating cartridges inserted into bores 401A and 401B of the lower plate 10 may likewise be controlled independently and, together with the fluid flow channels, be used to regulate the temperature of a substrate stage, a substrate, or any specimen disposed on the substrate. In some embodiments, the heating and / or cooling elements are each independently operated to as to maintain a predetermined temperature gradient between different portions of the upper and lower plates and the substrate stage.

[0283] FIG. 30C provides yet a further alternative embodiment of a chamber formed from upper and lower plates, where the upper and lower plates comprise a plurality of fluid flow channels 411A through 411F. FIG. 30D illustrates an embodiment where the lower plate 16 is similar to that depicted in FIG. 30B, but where the upper plate includes a thermoelectric module 402 to effectuate heating and / or cooling of the body 33 of the upper plate 30. In some embodiments, and as illustrated in FIGS. 30E and 30F, the fluid channels of the upper and lower plate 30 and 10, respectively, may be connected such that fluid flows through the fluid channels of one of the upper or lower plates and then flows through the fluid flow channels of the other of the lower or upper plates.

[0284] In some embodiments, any of the heating and / or cooling elements within the upper plate 30 may be operated in conjunction with those heating and / or cooling elements within the lower plate 10, or those heating and / or cooling elements 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, that the temperature of the substrate stage, any portion of a substrate supported by the substrate stage, and / or the chamber may be controlled.

[0285] For example, it is possible to independently operate the various heating and / or cooling elements present in the upper and lower plates such that at least a portion of the substrate, or the specimen disposed on the substrate, remains the coldest structure within the chamber, for example the substrate or the specimen disposed on the substrate are maintained at a temperature lower than the upper plate, the lower plate, ports, valves, and / or any other structure within the chamber formed from the upper and lower plates. In some embodiments, the heating and / or cooling elements are each independently operated to as to maintain a predetermined temperature gradient between different portions of the upper and lower plates and the substrate stage.

[0286] With reference to FIG. 40B, different temperature zones may be established, such as temperature zones A, B, C, D, and E. In some embodiments, each of these different temperature zones are adjacent to and / or in thermal communication with portions of the body 16 of lower plate 10 and / or the body 33 of upper plate 30. The temperature within each of these zones may be established, maintained, and / or regulated by independently controlling any number of heating and / or elements in thermal communication with any of the depicted zones, including heating and / or cooling elements disposed within any of 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 substrate stage 12C, or the substrate stage itself are maintained at a temperature less than other portions of the body 16 of the lower plate 10 or other portions of the body 33 of the upper plate 33. In some embodiments, at least a portion of the substrate disposed within the chamber formed from the upper and lower plates is maintained as the coolest components within the chamber, such as during an unmasking operation (see FIGS. 43B - 43G).

[0287] By way of example, and again with reference to FIG. 40B, zone A may be established at a temperature which is less than any of zones B, C, D, and / or E In some embodiments, zones A and B are maintained at about the same temperature, while zones C, D, and E are maintained at temperatures which are comparatively less. The skilled artisan will appreciate that one or more heating and / or cooling elements positioned proximal to zone B will allow zones A and B to maintained within a first temperature range, while one or more heating and / or cooling elements positioned proximal to each of zones C, D, and E will allow zones C, D, and to be maintained with 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 about 2% lower than the second temperature range. In other embodiments, the first temperature range is about 3% lower than the second temperature range. In some embodiments, the first temperature range is about 4% lower than the second temperature range. In some embodiments, the first temperature range is about 5% lower than the second temperature range. In some embodiments, the first temperature range is about 7% lower than the second temperature range. In some embodiments, the first temperature range is about 8% lower than the second temperature range. In some embodiments, the first temperature range is about 10% lower than the second temperature range. In some embodiments, the first temperature range is about 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.

[0288] FIGS. 41A, 41B, and 41C each illustrate a substrate 15 in thermal communication with one or more heating and / or cooling elements (see, e.g., the heating and / or cooling elements 401A, 401B, 411A, and 411B in FIG. 41B). Each FIGS. 41A, 41B, and 41C illustrate that individual heating and / or cooling elements may be independently operated such that at least a portion of the substrate (or a specimen disposed on the substrate) within a chamber formed by the upper and lower pates 30 and 10, respectively, may have a temperature which is less than a temperature of other components disposed within the chamber. In this regard, and as noted above, any condensation formed within the chamber may be driven to that coldest portion of the chamber, for example the coldest portion of the specimen or the substrate, during any temperature ramp-down operation. In this way, evaporated fluids and / or reagents may be returned to the substrate and / or the specimen disposed on the substrate.SPECIMEN PROCESSING

[0289] The specimen processing assemblies of the present disclosure are configured to facilitate the processing of specimens disposed on the surface of a substrate. In some embodiments, the processing of specimens includes unmasking a specimen, e.g. antigen retrieval and / or target retrieval.

[0290] In some embodiments, different specimen processing operations may take place in the same area or in different areas of the specimen processing assemblies. In some embodiments, each of these different specimen processing areas may be pre-designated areas, for example areas that are set aside with the specimen processing assembly for performing a specific processing operation. FIGS. 13A and 13B provide two non-limiting examples of pre-designated specimen processing areas in the context of a movable lower plate coupled to a sub-assembly 101. As described further below, the lower plate may be moved along the sub-assembly 101 to different pre-designated processing areas. By way of another example, one or more dispense devices may be positioned over one or more substrate trays or carousels, such as depicted in FIGS. 20A and 20B. In this example, the area defined by the one or more substrate trays, carousels, and dispense devices is a pre-established specimen preparation area.

[0291] Alternatively, specimen processing areas may be defined by the presence or absence of one or more specimen processing assembly components. For example, for a specimen processing assembly including a fixed lower plate, a user may position a specimen bearing substrate on an upper surface of a substrate stage of the fixed lower plate, and this would define a loading area. Then, one or more dispense devices, mixing devices, and / or liquid removal devices may be moved to that fixed lower plate to dispense one or more fluids and / or reagents to the substrate. The presence of the one or more dispense devices in this area would define the area as a specimen preparation area.

[0292] Subsequently, an upper plate (and / or a force generating member) may then be moved to the fixed lower plate (after the one or more dispense devices, mixing devices, and / or liquid removal devices are moved out of the area), such that a chamber may be formed between the movable upper plate and the fixed lower plate. The formed chamber would then define an unmasking area. As in this example, any one area serves multiple purposes, e.g. the same area may be used as a preparation area and an unmasking area depending on what devices and / or components are positioned in that area.

[0293] By way of another example, a lower plate may be moved from a first position to a second position. In some embodiments, the first position is in a loading area and thus constitutes a pre-designated area. In some embodiments, the second position may be defined as a preparation area or an unmasking area depending on what component is present with the lower plate. For example, one or more dispense devices may be moved to the lower plate in the second position thereby defining a preparation area. Following the dispensing of one or more fluids and / or reagents to a specimen bearing substrate, an upper plate may then be moved to the lower plate in the second position such that a chamber may be formed. In some embodiments, the formed chamber defines an unmasking area.

[0294] In the context of a specimen processing assembly 100 including a lower plate 10 movably coupled to a sub-assembly 101, the lower plate 10 may be moved from a first position, to a second position, to a third position, and to an n th< position. For example, in some embodiments, the lower plate 10 may be moved between a loading area, a preparation area, and an unmasking area. Of course, the lower plate 10 may be moved or held at any intermediate area in between any of the loading, preparation, or unmasking areas. In some embodiments, specimen bearing substrates are loaded onto a lower plate in a loading area. Alternatively, specimen bearing substrates are loaded onto an intermediary member for holding and / or transporting a substrate in a loading area.

[0295] Examples of intermediary members, such as substrate loaders 302, substrate holders 310, and / or substrate trays, are depicted in FIGS. 19A - 19C and 20A - 20B. In these embodiments, the intermediary member may transfer the substrate to the lower plate for further processing. Following the loading of the specimen bearing substrate, the specimen may be treated in a preparation area with one or more fluids and / or reagents. Unmasking operations may then be carried out at elevated temperatures and / or pressures in an unmasking area (see FIGS. 43A -43F). For example, once a specimen bearing substrate 15 is moved into position, such as within a chamber formed by the upper plate 30 and lower plate 10, an unmasking operation may allow surface antigens and / or nucleic acids to become visibly distinguishable, whether or not a subsequent staining procedure is applied thereto. Unmasking may be performed according to any of the methods described in PCT Publication No. WO / 2013 / 079606.

[0296] FIGS. 13A and 13B illustrate the various specimen processing areas in the context of a movable lower plate. In these embodiments, the lower plate 10 may be moved between a loading area 110, a preparation area 111, and an unmasking area 112. In some embodiments, the lower plate 10 may be moved or held at any intermediate area in-between any of the loading 110, processing 111, or unmasking areas 112.

[0297] In some embodiments, the lower plate 10 is initially moved to a loading area, such as illustrated in FIGS. 13A and 13B (if it is not already located within such an area). Once in the loading area, a specimen bearing substrate 15 may be positioned on the upper surface 12C of the substrate stage 12 of the lower plate 10, such as by an operator of the specimen processing assembly or a system including the specimen processing assembly. In some embodiments, the specimen bearing substrate 15 is a microscope slide.

[0298] Next, the lower plate 10 may be moved from the loading area 110 to one of a preparation area 111 or an unmasking area 112. In one embodiment, such as depicted in FIG. 13A, a lower plate 10 is moved from the loading area to the preparation area such that one or more fluids and / or reagents may be deposited or dispensed onto the surface of the substrate 15 and / or any sample disposed thereon. In some embodiments, one or more unmasking agents may be dispensed onto the specimen while in the preparation area, and unmasking may commence upon the dispensing of such reagents. In some embodiments, a total volume of one or more fluids and / or reagents dispensed to the substrate or the specimen disposed on the substrate ranges from between about 100µL to about 2000µL. In some embodiments, a total volume of one or more fluids and / or reagents dispensed to the substrate or the specimen disposed on the substrate ranges from between about 100µL to about 1500µL. In some embodiments, a total volume of one or more fluids and / or reagents dispensed to the substrate or the specimen disposed on the substrate ranges from between about 100µL to about 1250µL. In some embodiments, a total volume of one or more fluids and / or reagents dispensed to the substrate or the specimen disposed on the substrate ranges from between about 100µL to about 1000µL. In some embodiments, a total volume of one or more fluids and / or reagents dispensed to the substrate or the specimen disposed on the substrate ranges from between about 100µL to about 900µL. In some embodiments, a total volume of one or more fluids and / or reagents dispensed to the substrate or the specimen disposed on the substrate ranges from between about 150µL to about 800µL. In some embodiments, a total volume of one or more fluids and / or reagents dispensed to the substrate or the specimen disposed on the substrate ranges from between about 200µL to about 750µL. In some embodiments, a total volume of one or more fluids and / or reagents dispensed to the substrate or the specimen disposed on the substrate ranges from between about 250µL to about 700µL. In some embodiments, a total volume of one or more fluids and / or reagents dispensed to the substrate or the ...

Claims

1. A system for unmasking protein antigens and nucleic acid targets from fixed biological samples comprising: (i) at least one unmasking chamber being configured for heating and / or pressurizing a specimen disposed on a substrate (15) in a presence of one or more unmasking agents for a pre-determined amount of time, the unmasking chamber having a predetermined interior volume, wherein the at least one unmasking chamber comprises an upper plate (30) and a lower plate (10), wherein the lower plate (10) includes a lower engagement surface (11) and one or more substrate stages (12) adapted to hold the substrate (15) horizontally within the at least one unmasking chamber, and wherein the upper plate (30) includes an upper engagement surface (31); and wherein the upper plate (30) comprises one or more steam injection ports for introducing steam into the at least one unmasking chamber; wherein at least one of the lower plate (10) and the upper plate (30) further comprises at least one seal body; (ii) a staining module; and (iii) a steam reservoir; characterized in that: • the upper engagement surface (31) is complementary to the lower engagement surface (11); and • the upper plate (30) includes a cavity (32) recessed relative to the upper engagement surface (31).

2. The system of claim 1, wherein the upper plate (30) is coupled to a force generating member.

3. The system of claim 2, wherein the force generating member is selected from the group consisting of a motor, a spring, a screw, a lever, a piston, a cam, or any combination thereof.

4. The system of any one of claims 1 - 3, wherein the at least one unmasking chamber comprises a predetermined internal volume ranging from between about 14cm3 to about 25cm3.

5. The system of any one of claims 1 - 4, wherein at least one of the lower and / or upper plates (30) further comprises one or more alignment members (14).

6. The system of any one of claims 1 - 5, wherein at least one of the lower and / or upper plates (30) further comprise one or more temperature and / or pressure sensors.

7. The system of claim 1, wherein the at least one seal body is removable.

8. The system of claim 7, wherein the lower plate (10) comprises a groove (13) and wherein the at least one removable seal body at least partially engages the groove (13).

9. The system of claim 1, wherein the upper plate (30) and the lower plate (10) are removably couplable together such that upon reaching a predetermined internal pressure and / or temperature the coupling may be disengaged and the upper and lower plates (30, 10) rapidly separate from each other.

10. The system of claim 1, wherein at least one of the lower plate (10) and / or the upper plate (30) is in thermal communication with at least one heating and / or cooling element (21).

11. The system of claim 10, wherein the at least one heating and / or cooling element (21) is operable in conjunction with the one or more steam injection ports.

12. Use of a system of any one of claims 1 - 8 in the preparation of an unmasked specimen.

13. The use of the system of claim 12, wherein the specimen is a histology sample.

14. The use of the system of claim 12, wherein the specimen is a cytology sample.