WO2009085842A1

WO2009085842A1 - Relative-translational liquid application and removal

Info

Publication number: WO2009085842A1
Application number: PCT/US2008/087193
Authority: WO
Inventors: Vincent R. Rizzo; Roy Rizkovsky; Brian H. Kram; David Chafin; Ryan Reeser; Michael D. Tucker; Peter A. Riefenhauser; Lizhen Pang
Original assignee: Ventana Medical Systems, Inc.
Priority date: 2007-12-21
Filing date: 2008-12-17
Publication date: 2009-07-09
Also published as: WO2009086048A1

Abstract

An apparatus is disclosed for applying a liquid to a substantially flat substrate that takes advantage of the spreading of liquids within a capillary space to better cover a substrate but applies a motive force to the liquid in the capillary space to enhance mixing and redistribution in a manner that avoids shortcomings of prior methods. In one aspect, an apparatus is disclosed that includes a platen having one or more depressions around which a liquid entrained in a capillary space between the platen and a substrate will flow when relative motion between the platen and substrate is induced, thereby inducing mixing and/or redistribution of the liquid across the surface of the substrate facing the platen.

Description

RELATIVE-TRANSLATIONAL LIQUID APPLICATION AND

REMOVAL

Related Application Data This claims the benefit of U.S. Provisional Patent Application No. 61/015951, filed December 21, 2007 and U.S. Provisional Patent Application No. 61/015946, filed December 21, 2007, both of which applications are incorporated by reference herein.

Field

The present invention relates to a system and method for applying a liquid to a substantially flat substrate. More particularly, the present invention relates to a system and method for effectively mixing and redistributing a liquid that is confined within a capillary space while in contact with a substrate.

Background

Many reagents used to analyze biological samples are precious and expensive, and some reagents pose hazards during use and disposal. Thus, it is desirable to minimize the amount of reagent used in any particular treatment of the sample. For liquid reagents, the amount of reagent can be reduced by either reducing the concentration of reagent dissolved in a liquid or by reducing the volume of reagent utilized. When a certain concentration of reagent is required for a particular analysis or the reagent is a pure liquid, only the volume can be reduced. However, in the context of a biological sample(s) distributed on a substrate, use of a smaller volume of reagent may not be possible since the smaller volume might not completely cover the sample and therefore lead to analysis inconsistencies across the sample.

One way to increase the coverage of a sample is to spread the reagent across the substrate by creating a capillary space between a flat surface of the substrate and a second, opposing surface. A liquid confined to such a capillary space will tend to spread and fill the space due to capillary forces, thereby better covering the flat surface of the substrate and any sample placed thereon. Unfortunately, once a liquid has spread to fill a capillary space, further motion of the liquid within the capillary space is restricted by capillary forces. Passive mixing and redistribution of a liquid confined to a capillary space often are slow or non-existent. If a reagent dissolved in the liquid is consumed by reaction or binding to a sample mounted on the substrate, a zone of depletion of the reagent will form around the sample. Unless the reagent is replenished within this depletion zone around the sample by mixing, redistribution, or exchange of the liquid reagent in the space for fresh reagent, consumption of the reagent by the sample will slow and extend the time needed to accomplish the analysis. Furthermore, concentration differences that develop toward the edges of the zone of depletion can lead to inhomogeneous treatment across the sample and result in undesirable effects such as staining gradients.

In order to overcome the constrained mixing and redistribution of a liquid within a capillary space, mixing and redistribution are typically accomplished by applying a motive force to the liquid. Some systems and methods mix and redistribute liquids by pumping them into and out of the capillary chamber through one or more ports. Others mix by altering the dimensions of the capillary chamber to induce flow within the chamber. Systems and methods for moving liquids within a capillary space by physical alteration of the capillary chamber include those disclosed in U.S. Patent Application Publication No.20030157503, which describes a flexible cover used to form a capillary chamber over a sample on a substrate, and a roller mechanism that deforms the cover inward as it moves across the cover. Schermer et al (U.S. Patent No. 6,485,918) describes a substantially rigid lid and a gasket that deforms more easily than the lid. Actuators apply forces to the cover and deform the gasket of the cover, and when the actuators produce different forces the lid tilts toward one exerting a greater force, thus producing a flow of liquid reagent over the substrate. Schembri (EP 0891811 B 1 ) describes a capillary mixing mechanism that moves the inner face of at least one surface relative to the inner face of another, opposed surface to induce mixing of a liquid within a thin chamber. Particular embodiments include a flexible surface that moves in response to a series of rotational forces to repeatedly bulge out and return to its original shape thereby forcing the liquid to redistribute across the chamber. Other embodiments disclosed by Schembri include a compression inducing mechanism, a tension inducing mechanism and a shear inducing mechanism, each of which can be used for mixing of a liquid between two rigid materials by continuously or intermittently moving the rigid materials up and down or side-to-side. The tension inducing mechanism of Schembri is disclosed to pull one material away from the other material by mechanical, magnetic or vacuum attachment causing liquid to move toward portions of the capillary space that are not expanded by the tension force. Release of the tension force causes a liquid to move back toward the previously expanded portion of the liquid chamber and impart mixing in the chamber. What is still needed is a sample treatment apparatus and method that can provide more controllable and homogeneous addition, mixing, redistribution and removal of liquids in a capillary gap. A system that is configurable, flexible and can be easily adapted to perform multiple sample treatment protocols (such as primary and special staining protocols, IHC and ISH) in a readily automated fashion also is desirable.

Summary

An apparatus is disclosed for applying a liquid to a substantially flat substrate that takes advantage of the spreading of liquids within a capillary space to better cover a substrate but applies a motive force to the liquid in the capillary space to enhance mixing and redistribution in a manner that avoids shortcomings of prior methods. The apparatus imparts a motive force to a liquid within a capillary space in a simple manner that is readily automated because it can impart such a force directly through the substrate itself, without the need for any type of specialized cover, gasket or means to impart motion to a separate cover. Clips and the like that are used to hold a substrate in prior devices can be avoided, reducing the likelihood that wicking pathways that draw precious liquids away from a surface of a substrate to be treated will be established. In one aspect, an apparatus is disclosed for applying a liquid to a substantially flat substrate. The apparatus includes a platen comprising a substantially flat surface and one or more depressions formed in the surface of the platen. A translator induces relative motion between the platen and the substrate and a liquid that is entrained within a capillary space between the flat surface of the platen and a flat surface of the substrate moves around the one or more depressions during the relative motion. Liquid is removed from contact with and then re-contacted with at least a portion of the flat surface of the substrate because the liquid substantially remains in the capillary space and does not enter the depressions. In a particular embodiment, alternating back and forth translation of a substrate relative to the platen induces sufficient motion of a liquid within the capillary space that the liquid mixes and is redistributed across the substrate's surface facing the platen. In a more particular embodiment, a biological sample to be treated with a liquid is adhered to the surface of the substrate facing the platen.

In another aspect that takes advantage of the benefits of the disclosed apparatus, an automated system is disclosed for treating a plurality of substantially flat substrates with a liquid. The system includes a plurality of single substrate treatment modules, each of which includes a single substrate treatment unit where the unit comprises the apparatus described above. Automation is simplified in the system particularly where forces used to induce relative motion and mix/redistribute a liquid within the capillary space are applied directly to the substrate, because in this case, additional automation required to handle a cover or gasket is avoided, as is disposal thereof. Furthermore, since a substrate can be simply placed on a platen, the substrate can easily be automatically loaded into a substrate treatment module without user intervention. Also included in the system are a liquid delivery system and a computer that controls the plurality of single substrate treatment units and the liquid delivery system according to a schedule for treatment of the plurality of substrates with the liquid.

In yet another aspect, a method is disclosed for applying a liquid to a substantially flat substrate. The method includes introducing the liquid into a capillary space between the substrate and a platen where the platen having a substantially flat surface and one or more depressions formed in the flat surface of the platen. The method also includes moving the substrate and the platen relative to one another such that during such relative motion the liquid in the capillary space substantially moves around the one or more depressions and becomes redistributed across a surface of the substrate.

The foregoing aspects, features and advantages of the disclosed apparatus, system and method are further illustrated in the drawings and detailed description that follow.

Brief Description of the Drawings

FIG. 1 is perspective diagram of an embodiment of a single substrate treatment unit.

FIG. 2 is a series of diagrams illustrating alternative curved depressions in a platen surface. FIG. 3 is a series of diagrams illustrating alternative polygonal depressions in a platen surface

FIG. 4 is a perspective diagram of an embodiment of a single substrate treatment unit incorporating a substrate sealing mechanism, a platen washing mechanism, and a sealing mechanism washing trough. FIG. 5 is a perspective diagram of an embodiment of a single substrate treatment unit incorporating a platen washing mechanism.

FIG. 6 is a perspective diagram of an embodiment of a single substrate treatment module.

FIG. 7 is another perspective diagram of an embodiment of a single substrate treatment module. FIG. 8 is an exploded, perspective diagram of an embodiment of a single substrate treatment module.

FIG. 9 is a perspective diagram of a system for treatment of a plurality of substrates in individual single substrate treatment modules. FIG. 10 is a perspective diagram of an embodiment of a reagent pack that can be utilized in a system for treating a plurality of substrates in individual single substrate treatment modules.

Detailed Description of Several Illustrative Embodiments The following description of several embodiments describes non-limiting examples that further illustrate the invention. All titles of sections contained herein, including those appearing above, are not to be construed as limitations on the invention, but rather they are provided to structure the illustrative description of the invention that is provided by the specification. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one skilled in the art to which the disclosed invention pertains. The singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a liquid" refers to one or more liquids, such as 2 or more liquids, 3 or more liquids, or even 4 or more liquids.

A "substantially flat substrate" refers to any object having at least one substantially flat surface, but more typically to any object having two substantially flat surfaces on opposite sides of the object, and even more typically to any object having opposed substantially flat surfaces, which opposed surfaces are equal in size but larger than any other surfaces on the object. A substantially flat substrate can be formed of any material, including a glass, silicon, a semiconductor material or a metal. Particular examples of substantially flat substrates include microscope slides (both 1" x 3" slides and 25mm x 75 mm slides), SELDI and MALDI chips, and silicon wafers. A "biological sample" refers to any sample obtained from, derived from or containing any organism including a plant, an animal, a microbe or even a virus. Particular examples of biological samples include tissue sections, cytology samples, sweat, tears, urine, feces, semen, pre-ejaculate, nipple aspirates, pus, sputum, blood, serum, tissue arrays, and protein and nucleic acid arrays. A "liquid" refers to any substance in a fluid state having no fixed shape but a substantially fixed volume. Examples of liquids include solvents and solutions. A liquid can be polar or non-polar, organic or inorganic, volatile or non-volatile, high viscosity or low viscosity, an emulsion or a true solution. Examples of solvents include water, alcohols, polyols, hydrocarbons and ionic liquids. Examples of solutions include aqueous solutions of a dye, a protein (such as an antibody), a nucleic acid (such as a hybridization probe), a buffer, an acid, a base or a salt. Other examples of solutions include mixtures of two or more solvents. Solutions also can include neutral proteins (such as albumin), detergents, proteases, protease inhibitors, nucleases, nuclease inhibitors, formamide, anti-microbial agents and the like that improve detection of analytes in a sample(s) and/or reduce non-specific or background interactions.

In one aspect, an apparatus is disclosed for applying a liquid to a substantially flat substrate. The apparatus includes a platen comprising a substantially flat surface and one or more depressions formed in the surface of the platen. The apparatus also includes a translator configured to induce relative motion between the platen and the substrate. Liquid entrained within a capillary space between the flat surface of the platen and a flat surface of the substrate substantially remains in the capillary space and moves around the one or more depressions during the relative motion, and in the process liquid is removed from contact with and then re-contacted with at least a portion of the flat surface of the substrate. The apparatus can further include a liquid applicator configured to deliver the liquid to the capillary space either directly, or by dispensing the liquid onto the platen and then moving the substrate to where the liquid is dispense, which applicator can be one or more of an aperture through the platen, a stationary or moveable nozzle, and a robotic dispenser. The platen of the apparatus can be made from any material, but is typically made of a metal, glass or a plastic, and can be coated or otherwise treated to affect its contact angle with a liquid or liquids applied using the apparatus. Choice of material and/or coating can be made to enhance the durability or the ease of renewal or cleaning of the surface. The platen can include a heater such that the temperature of a liquid in contact with its surface can be raised and maintained at a particular temperature, or it can include a device that can both heat and cool a liquid in contact with the surface (such as a Peltier device or thermal liquid conduits). More than one heater or cooling device can be included in the platen, and multiple such platens can be heated or cooled simultaneously or independently. In one embodiment, the platen further comprises at least two spacers (such as rails, although the liquid can itself can function as a single spacer in other embodiments) that hold the substrate and the substantially flat surface of the platen in spaced separation. The platen can further include one or more of a fluid phase trap and a stripping element as disclosed in co- pending U.S. Patent Application Publication No.20080102006, which publication is incorporated by reference herein. Briefly, a fluid phase trap is generally a shallow trough into which a liquid can be dispensed that aids in smooth filing of the capillary space between a substrate and a platen. A fluid phase trap also helps ensure that no bubbles are entrained into the liquid as it flows into the capillary space from the fluid phase trap. A stripping element is a combination of an air gap and an intersecting gap that intersects with the capillary space and provides a conduit through which a liquid will tend to move away from the substrate as the liquid encounters the air gap. A stripping element can include a capillary intersecting gap that passively removes liquid from a substrate. Stripping elements are more effective at removing liquids from a substrate than an air gap alone. Depressions can be molded or machined into the surface of the platen by any method, and the depressions can be made to be any depth that is sufficiently greater than the height (or width depending on orientation) of the capillary space such that during relative motion between the platen and the substrate the liquid remains in the capillary space and moves around the one or more depressions in the platen's surface. In particular embodiments, the depth of the depressions is at least 2 times the height of the capillary space, for example, at least 3 times the height of the capillary space or even at least 4 times the height of the capillary space. The depressions can be sharply defined in the surface or they can be radiused and or polished. In some embodiments, a depression can have a perimeter of which at least a portion is curved, for example, at least a portion of which comprises a circular perimeter, a hyperbolic perimeter, an elliptical perimeter or a parabolic perimeter. In other embodiments, a depression can have a perimeter, at least a part of which comprises a polygonal perimeter. In more particular embodiments, a depression can have a parabolic or hyperbolic perimeter, the axis of which parabolic or hyperbolic perimeter extends across at least a portion of a path of relative motion between the substrate and the substantially flat surface of the platen. In other more particular embodiments, the platen includes a pair of parabolic or hyperbolic depressions having a common axis and extending toward each other from opposite edges of the platen such that the liquid moves between apexes of the pair of depressions. Advantageously in this more particular embodiment, a vacuum hole can be located between the apexes of the pair of depressions and substantially along their common axis, such that moving the substrate past the depressions (or vice versa, or both) directs the liquid toward the vacuum hole and facilitates removal thereof through the vacuum hole.

In other particular embodiments, one or more depressions are configured such that as the substrate is moved relative to the depression(s) liquid is removed from contact with and then re-contacted with greater than 50% of the flat surface of the substrate, for example, greater than 60%, greater than 70%, greater than 80% or even greater than 90%. In still other particular embodiments, as the liquid moves substantially around at least one depression the liquid is moved past a vacuum port formed in the platen. In further embodiments, the platen comprises at least two depressions, between which depressions the liquid moves during the relative motion. A vacuum port can be located between the depressions to capture liquid as it moves between the depressions. In still further embodiments, the platen comprises at least two depressions and a flat space on the platen between the at least two depressions forms at least one capillary path along which the liquid flows as it flows around the at least two depressions, for example, more than 2 depressions and flat spaces on the platen between the depressions can form a plurality of capillary paths along which the liquid flows as it flows around the more than 2 depressions. In some embodiments of the apparatus, a biological sample is adhered to the substrate on a surface facing the platen. In particular embodiments, the sample is a tissue section or a cytology sample.

In other embodiments, relative motion between the substrate and the platen is oscillatory or bidirectional, back and forth past the depressions. Any number of pauses of any particular length can be included in a pre-determined series of motions of the substrate relative to the platen. The speed of relative motion also can be varied as can the number of times the substrate is moved past a depression or depressions to re-distribute/mix the liquid. The flat surface of the platen and a flat surface of the substrate are typically parallel, and the combination can be oriented at any angle between horizontal and vertical. However, in particular embodiments, the platen is horizontal and on bottom, and the substrate is horizontal and on top. Either or both of the platen or substrate can move during relative motion, but more typically, the substrate is moved by the translator and the platen is stationary. In some embodiments, the translator that induces relative motion between the substrate and the platen comprises one or more of a belt drive, a screw drive, a chain drive, and a slide drive. It also should be noted that the apparatus described above can be operated manually or automatically.

In another aspect, an automated system is disclosed for treating a plurality of substantially flat substrates with a liquid. The system includes a plurality of single substrate treatment modules where each module comprises a single substrate treatment unit. The single substrate treatment unit includes a platen comprising a substantially flat surface and one or more depressions formed in the surface of the platen. A translator is included that is configured to induce relative motion between the platen and the substrate. A liquid is entrained between the flat surface of the platen and the substrate within a capillary space, and the liquid substantially moves around the one or more depressions during the relative motion such that the liquid is removed from contact with a portion of the substrate and is then subsequently reapplied to the portion. The system also includes a liquid delivery system and a computer that controls the plurality of single substrate treatment units and the liquid delivery system according to a schedule for treatment of the plurality of substrates with the liquid. Each single substrate treatment module can include a chamber that can enclose at least a portion of the single substrate treatment unit such that the environment (such as temperature, humidity and pressure) of the unit can be controlled, for example, to reduce evaporation of liquids applied to the substrate. Alternatively, the entire single substrate treatment module can be enclosed in a chamber.

The system also can optionally include one or more of a substrate transporter, a substrate drying unit, a substrate holding cassette, a machine-readable code reader, a source of vacuum, a source of pressurized gas, a waste-handling system and a reagent transporter.

In a particular embodiment, the liquid delivery system comprises a dedicated robotic dispenser in each of the plurality of single substrate treatment modules. Also in particular embodiments, the liquid delivery system can comprise a multi-well reagent pack and a reagent pack delivery system. For example, the reagent pack delivery system can comprises a robotic delivery system that is configured to move a single reagent pack between more than one single substrate treatment modules so that each module does not require a separate reagent pack delivery system. In order to coordinate the delivery of liquids and the treatment of substrates under computer control, one or both of the substrate and a reagent container can be labeled with a machine-readable code. Examples of machine-readable codes that can be used include linear barcodes (such as code 128), multi-dimensional barcodes (such as optical characters, data matrices and infoglyphs), RFID tags, Bragg-diffraction gratings, magnetic stripes, or nanobarcodes (such as spatial and spectral patterns of fluorescent nanoparticles or spatial patterns of magnetic nanoparticles). The liquid delivery system also can include a plurality of nozzles in each of the single substrate treatment modules, where the plurality of nozzles connected to a plurality of bulk reagent supplies.

The disclosed single substrate treatment module can include an interchangeable single substrate treatment unit, and it is also possible to include one or more additional single substrate treatment modules where the additional substrate treatment modules comprise one or more of the disclosed single substrate treatment unit and one or more of any other type of single substrate treatment unit. For example, an alternate single substrate treatment unit can include a substantially flat liquid application surface and a spacer that holds the substrate and the liquid application surface in spaced separation to form a capillary space. A liquid that is introduced into the capillary space is moved within the space by at least two separators disposed on different (such as opposite) sides of the liquid application surface, wherein the separators contact a surface of the substrate facing the liquid application surface and move the substrate away from the liquid application surface. In one embodiment, the substrate rests on top of the spacers and above the flat liquid application surface. The separators on opposite sides of the liquid application surface can contact a lower surface of the substrate outside of the capillary space, thereby avoiding contact with the liquid that could initiate wicking flow of the liquid out of the capillary space. The separators impart a motive force upward to move the substrate away from the liquid application surface, thereby altering the capillary space to induce liquid movement within the space. In a particular embodiment, alternating application of lifting forces to opposite sides of a substrate causes a back and forth motion of a liquid within the capillary space that mixes and redistributes the liquid across the substrate's lower surface.

It is not only is it possible to interchange a single substrate treatment unit with an alternate single substrate treatment unit in a given module, but it also is possible to exchange entire modules. Having replaceable and interchangeable units in the modules and exchangeable modules makes servicing and re-configuring the system simpler and quicker.

Thus, in yet another aspect, a system for handling and treating substantially flat substrates with a liquid is disclosed. The system includes a plurality of single slide treatment modules, each module comprising a single substrate treatment unit and a separate robotic liquid dispenser. Also included in the system is a reagent pack delivery system configured to deliver a reagent pack to each of the plurality of single slide treatment modules. The entire system further includes a processor that controls the plurality of single substrate treatment units, the separate robotic liquid dispensers, the reagent pack delivery system and other system components (such as bulk liquid delivery systems) to perform a pre-determined sequence of treatment steps on the substrates. Each substrate can be treated independently with the same or different sequence of treatment steps. The reagent pack can hold a single reagent and can be delivered to different treatment modules according to a schedule when they are required for a particular step in the treatment of a substrate. Otherwise, the reagent pack can be stored, for example, in a refrigerated location. Alternatively, the reagent pack can include a plurality of different reagents needed to treat a substrate according to a single multi-step treatment protocol.

In yet another aspect, a method is disclosed for applying a liquid to a substantially flat substrate. The method includes introducing the liquid into a capillary space between the substrate and a platen, the platen having a substantially flat surface and one or more depressions formed in the flat surface of the platen and moving the substrate and the platen relative to one another such that during such relative motion the liquid in the capillary space substantially moves around the one or more depressions and becomes redistributed across a surface of the substrate. In some embodiments, moving the substrate and the platen relative to one another comprises moving the substrate relative to a stationary platen. In other embodiments, moving the substrate and the platen relative to one another comprises moving in a first direction and moving in a second direction, for example the first and second directions can be opposite directions. In a particular embodiment, moving in first and second, opposite directions is repeated at least 2 times.

FIG 1 shows a particular working embodiment of a single substrate treatment unit 10 according to the disclosure that can be used to practice the disclosed method. In this embodiment, platen 12 includes a curved depression 14 and spacers 16, which are in this case rails integral with platen 12. A vacuum hole 17 is located near the apex of the curved depression 14. Substrate 40 rests on rails 16 and is moved across platen 12 and past depression 14 by transporter 18 (a slide-drive in this case). Transporter 18 is moved by stepper motor 20 which is connected to transporter 18 through a screw drive 22. As stepper motor 20 turns in one direction it pushes the transporter away taking substrate 40 along rails 16. When it turns in the other direction it draws the transporter back and brings substrate back along the rails. In operation, a liquid introduced to the capillary space between substrate 40 and the substantially flat surface of platen 12 will spread out under the substrate. When substrate 40 is moved past depression 14 the liquid will move around the apex of depression 14, substantially staying within the capillary space except over the depression. The flow of liquid around the depression induces mixing within the liquid, and redistributes the liquid on the lower surface of substrate 40 by removing the liquid from contact with a portion of the substrate that is over the depression and then subsequently re-contacting that portion of the substrate when the liquid moves back under the substrate after having gone around the depression. Removal of liquid from the capillary space can be enhanced if the motion of the substrate relative to the platen is combined with application of a vacuum at vacuum port 17. As the substrate reaches depression 14 the liquid in the capillary space flows toward vacuum port 17, and if it is removed through the vacuum port, the combined translation past the depression and removal through the vacuum port effectively "wipes" and removes the liquid from under the substrate.

FIG. 1 shows only one possible configuration of depression 14. FIGS. 2A-2H show additional examples of curved depression configurations and combinations thereof that can be utilized for different treatment protocols. Likewise the diagrams of FIG. 3 show examples of polygonic depression patterns that can be used for other treatment protocols. In FIGS 2A-2H, and 3, reference numbers are the same as shown in FIG. 1.

FIG. 4 shows another embodiment of a single substrate treatment unit that includes additional features that are useful for high temperature substrate treatment steps and for cleaning the platen surface between treatment steps or between treatments of different substrates using the unit. In FIG. 4, platen 12 is barely visible under platen cleaning unit 50, which also includes cleaning/rinse liquid supply 52 and vacuum line 54. In typical operation, a cleaning liquid would be caused to flow from supply 52 under cleaning unit 50 and across the platen surface to vacuum line 54 where it would be removed. Cleaning unit 50 can be automatically positioned over the platen on either side of a central depression (like that shown in FIG.1 ) with motor 56 and positioning mechanism 57 so that both sides of the platen can be cleaned, for example, while a substrate is undergoing an incubation step on the opposite side. Substrate 40 is shown under sealing unit 60 which surrounds and seals a liquid in the capillary space under the substrate. Sealing unit 60 typically includes a compressible seal around its lower perimeter and can be automatically moved from the position shown to within cleaning trough 62 by positioning mechanism 63 powered by motor 64. Placement of sealing unit into a cleaning liquid contained within cleaning trough 62 helps ensure against cross-contamination of successive substrates that are treated on the unit and helps prevent cross-contamination of reagents successively applied to a single substrate. The sealing unit is an example of a chamber that can enclose at least a portion of the single substrate treatment unit (in this case the portion where the substrate is located) in order to limit evaporation of liquid from the capillary space between the substrate and the substantially flat surface of the platen. When sealing unit 60 and cleaning unit 50 are both moved away from platen 12, substrate 40 can be moved across the platen by translator 18.

FIG. 5 is yet another embodiment of a single substrate treatment unit 10 that includes only the platen cleaning unit. All reference numbers in FIG 5 correspond to those utilized in FIG. 4, except that without the sealing unit included it is possible to see depression 14 in platen 12. FIG. 6 shows a cutaway perspective view of an embodiment of a single substrate treatment module 100. Within module 100 are seen single substrate treatment unit 10, dedicated robotic pipettor 102, bulk liquid supply 104 and reagent mixing and pipettor washing station 106. Bulk liquid supply 104 can be seen plumbed to a plurality of liquid delivery wells of single substrate treatment unit 10. During operation, dedicated robotic pipettor 102 can retrieve one or more liquids from a reagent delivery pack (such as by piercing a cover over a well of a multi-well reagent pack) delivered to the module 100 by a reagent pack delivery system (as shown in FIG. 9). The liquid can either be delivered to the capillary space of the treatment unit 10 or can be added to a vial in the mixing and washing station 106, and a second liquid to be mixed can also be added and the two mixed, then aspirated and delivered to treat a substrate on the treatment unit 10. Between retrievals of liquids, the tip of the robotic pipettor 102 can be washed in a vial of the washing station 106 that contains a wash solution. FIG. 7 is an alternative view of the embodiment of the single substrate treatment module 100 of FIG. 6. Again, treatment unit 10 and bulk liquid supply 104 are shown, as are a syringe pump assembly 108 for dispensing liquids and a valve assembly 110 for providing vacuum to the robotic pipettor during aspiration of a liquid. FIG. 8 is an exploded view of the components discussed in FIGS. 6 and 7, wherein the reference numbers are the same as above. Cutouts 112 in the housing of the module 100 provide access to the module by a reagent pack delivery system (shown in FIG. 9). Treatment unit 10 is interchangeable.

FIG. 9 is a perspective view of an embodiment of a system for simultaneously treating a plurality of substrates with one or more liquids, according to the same or different treatment protocols. This embodiment also includes features that permit singulation of substrates input into the system (such as microscope slides bearing tissue samples) for processing according to pre-determined protocols and for retrieving substrates treated in the system either singly or sorted into some pre- determined grouping (such as same patient samples, same type of treatment, same responsible person such as a pathologist). The system also includes the use of multi- well reagent packs that have a top portion having the same dimensions as the substrates handled by the system (such as the dimensions of a microscope slide), making it possible to move substrates and reagent packs easily with the same type of robotic gripper.

System 200 of FIG. 9 includes a reagent delivery system that delivers reagent packs to single substrate treatment modules 100. The reagent delivery system in this system includes a reagent handling robot 204 that removes a multi-well reagent pack 206 from reagent carousel 202 (which holds a plurality of reagent packs and can be refrigerated), and places the reagent pack 206 onto one or more reagent conveyors

208 that each serve different sets of treatment modules 100, such as on different levels as shown. Conveyors 208 pass through cutouts (112 of FIG. 8) in the treatment modules 100 and can stop inside of the different modules on a give level such that a dedicated robotic pipettor within the module can access the reagent packs. Substrates 40 are moved within the system by substrate handling robot 216, which shuttles substrates between slide cassettes 212, drying oven 214, treatment modules 100 and any number of other system components that can be included. Movement of substrates and reagent packs within the system and the treatment of the substrates is computer controlled according to a pre-determined schedule, that can be interrupted if necessary to expedite treatment of a given substrate (or sample adhered thereto) or retrieve samples if there is a system malfunction. Also shown in FIG. 8 is bulk liquid supply 210 that is plumbed to the substrate treatment modules. Treatment modules 100 be exchanged between positions within system 200, or additional modules can be added or some modules removed. Thus, in some embodiments, backplane 218 will further include multiple common electrical, fluidic, vacuum and communication connections.

Although not explicitly shown in FIG. 9, it is to be understood that the system can further include additional components typically found on substrate treatment systems. For example, such a system will typically include one or more power supplies, data and electrical connections, a waste handling system (such as a waste collection vessel plumbed to the vacuum ports of the treatment units of the treatment modules), a source of vacuum, a source of pressurized gas, sensors for detecting temperature or location of components or for keeping track of substrates labeled with machine-readable codes (e.g. machine-readable code readers), additional substrate handling robots, additional ovens, incubation chambers and the like, and any number of control units that control individual components or the system. For example, a control unit (such as a microprocessor, microcomputer or computer) controls pumps, motors and valves to coordinate substrate movement within the system, delivery of reagents within the system, dispensation of liquids to the single substrate treatment units, sorting of substrates etc. The control unit also can have stored in memory alternate sets of commands to enable a variety of treatment staining protocols, or can interact with a user through a graphical user interface so that a user can develop a new protocol suitable for any particular treatment procedure. A control unit can further be connected to a large network of computers such as a Laboratory Information System (LIS) or any other type of system utilized to track patient samples and monitor workflow in a laboratory (see, for example, U.S. Patent Application Publication Nos. 2007/196909 and 2005/159982). The control unit also can be configured to provide remote monitoring and trouble-shooting of the instrument. In a particular embodiment, a unique identifier is associated with a particular sample adhered to a particular substrate and that particular sample can be tracked and associated with other similar samples throughout a laboratory. Furthermore, a single control unit could be connected to multiple substrate treatment systems, each of which can include a plurality of individual substrate treatment modules.

FIG. 10 is a perspective drawing of a reagent pack 206 that can be used in the disclosed system. Reagent pack 206 includes a top portion 300 and a bottom portion 302. Top portion 300 has a size and thickness that is substantially the same as the size and thickness of a substrate that is treated by the disclosed system, such as the same size and thickness of a microscope slide. Bottom portion 302 extends below and is integral with top portion 300. Bottom portion 302 is smaller in size but is thicker than top portion 300. One or more reagent wells 304 extend through top portion 300 and into, but not through, bottom portion 302. The one or more reagent wells 304 can be shaped and sized in dimensions similar to the wells in a typical 96-well microtiter plate. A foil or thin plastic covering can be applied over the one or more wells to protect reagents contained therein, and such covering also can be thin enough to be pierced by a robotic dispenser tip such that a reagent in a given well is protected until it is retrieved by the dispenser for use.

It is to be understood that the disclosed invention is not limited to the particular embodiments illustrated above and that many changes may be made without departing from the true scope and spirit of the invention, which is defined by the claims that follow. Furthermore, those skilled in the art to which the invention pertains will recognize, or be able to ascertain through no more than routine experimentation, many equivalents to the embodiments described herein. Such equivalents are intended to fall within the scope of the claims.

Claims

We Claim:

1. An apparatus for applying a liquid to a substantially flat substrate, comprising: a platen comprising a substantially flat surface and one or more depressions formed in the surface of the platen; and a translator configured to induce relative motion between the platen and the substrate; wherein the liquid is entrained within a capillary space between the flat surface of the platen and a flat surface of the substrate, and wherein the liquid substantially remains in the capillary space and moves around the one or more depressions during the relative motion such that the liquid is removed from contact with and then re-contacted with at least a portion of the flat surface of the substrate.

2. The apparatus of claim 1 , wherein the platen further comprises at least two spacers that hold the substrate and the substantially flat surface of the platen in spaced separation.

3. The apparatus of claim 1, wherein the one or more depressions comprise one or more depressions having a perimeter of which at least a portion is curved.

4. The apparatus of claim 3, wherein the one or more depressions having a curved perimeter comprise one or more depressions that have a perimeter, at least a part of which, comprises a circular perimeter, a hyperbolic perimeter, an elliptical perimeter or a parabolic perimeter.

5. The apparatus of claim 1 , wherein the one or more depressions comprise one or more depressions having a perimeter, at least a part of which, comprises a polygonal perimeter.

6. The apparatus of claim 1, wherein the one or more depressions comprise one or more depressions having a parabolic or hyperbolic perimeter, the axis of which parabolic or hyperbolic perimeter extends across at least a portion of a path of relative motion between the substrate and the substantially flat surface of the platen.

7. The apparatus of claim 1, wherein the one or more depressions are configured such that liquid is removed from contact with and then re-contacted with greater than 50% of the flat surface of the substrate.

8. The apparatus of claim 1 , wherein as the liquid moves substantially around at least one depression the liquid is moved past a vacuum port formed in the platen.

9. The apparatus of claim 1, wherein a biological sample is adhered to the substrate on a surface facing the platen.

10. The apparatus of claim 1 , wherein the platen comprises at least two depressions, between which depressions the liquid moves during the relative motion.

11. The apparatus of claim 10, wherein a vacuum port is located between the at least two depressions.

12. The apparatus of claim 1 , wherein the platen comprises a pair of parabolic or hyperbolic depressions having a common axis and extending toward each other from opposite edges of the platen such that the liquid moves between apexes of the pair of depressions.

13. The apparatus of claim 12, wherein a vacuum hole is located between the apexes of the pair of depressions and substantially along the common axis.

14. The apparatus of claim 1 , wherein the relative motion is oscillatory.

15. The apparatus of claim 1 , wherein the platen comprises at least two depressions and a flat space on the platen between the at least two depressions forms at least one capillary path along which the liquid flows as it flows around the at least two depressions.

16. The apparatus of claim 1, comprising more than 2 depressions and flat spaces on the platen between the depressions form a plurality of capillary paths along which the liquid flows as it flows around the more than 2 depressions.

17. The apparatus of claim 1, wherein the platen and the substrate are substantially horizontal.

18. The apparatus of claim 17, wherein the platen is on bottom and the substrate is on top.

19. The apparatus of claim 1 , wherein the substrate is moved by the translator and the platen is stationary.

20. The apparatus of claim 1, wherein the platen further includes one or more of a fluid phase trap and a stripping element.

21. The apparatus of claim 1 , wherein the transporter comprises one or more of a belt drive, a screw drive, a chain drive, and a slide drive.

22. The apparatus of claim 1, further comprising a liquid applicator configured to deliver the liquid to the capillary space.

23. The apparatus of claim 22, wherein the liquid applicator comprises one or more of an aperture through the platen, a stationary or moveable nozzle, and a robotic dispenser.

24. An automated system for handling and treating a plurality of substantially flat substrates with a liquid, comprising: a plurality of single substrate treatment modules; each module comprising a single substrate treatment unit; the single substrate treatment unit including a platen comprising a substantially flat surface and one or more depressions formed in the surface of the platen and a translator configured to induce relative motion between the platen and the substrate; wherein the liquid is entrained between the flat surface of the platen and the substrate within a capillary space, and the liquid substantially moves around the one or more depressions during the relative motion such that the liquid is removed from contact with a portion of the substrate and is then subsequently re- applied to the portion; a liquid delivery system; and a computer that controls the plurality of single substrate treatment units and the liquid delivery system according to a schedule for treatment of the plurality of substrates with the liquid.

25. The system of claim 24, further including one or more of a substrate transporter, a substrate drying unit, a substrate holding cassette, a machine-readable code reader, a source of vacuum, a source of pressurized gas, a waste-handling system and a reagent transporter.

26. The system of claim 24, wherein the liquid delivery system comprises a dedicated robotic dispenser in each of the plurality of single substrate treatment modules.

27. The system of claim 24, wherein the liquid delivery system comprises a multi-well reagent pack and a reagent pack delivery system.

28. The system of claim 27, wherein the reagent pack delivery system comprises a robotic delivery system configured move a single reagent pack between more than one single substrate treatment module.

29. The system of claim 24, wherein one or both of the substrate and a reagent container are labeled with a machine-readable code.

30. The system of claim 24, wherein the liquid delivery system comprises a plurality of nozzles in each of the single substrate treatment modules, the plurality of nozzles connected to a plurality of bulk reagent supplies.

31. The system of claim 24, further comprising one or more additional single substrate treatment modules; the additional single substrate treatment modules comprising an alternate single substrate treatment unit; the alternate single substrate treatment unit comprising a substantially flat liquid application surface against which the substrate is disposed, a spacer that holds the substrate and the liquid application surface in spaced separation to form a capillary space between the substrate and the liquid application surface into which the liquid is introduced, and at least two separators disposed on opposite sides of the liquid application surface, wherein the separators contact a surface of the substrate facing the liquid application surface and move the substrate away from the liquid application surface.

32. The system of claim 24, wherein single substrate treatment units are interchangeable between the plurality of singles substrate treatment modules.

33. The system of claim 31 , wherein the single substrate treatment unit and the alternate single substrate treatment unit are interchangeable between a single substrate treatment module and an additional single substrate treatment module.

34. A method for applying a liquid to a substantially flat substrate, comprising: introducing the liquid into a capillary space between the substrate and a platen, the platen having a substantially flat surface and one or more depressions formed in the flat surface of the platen; and, moving the substrate and the platen relative to one another such that during such relative motion the liquid in the capillary space substantially moves around the one or more depressions and becomes redistributed across a surface of the substrate.

35. The method of claim 34, wherein moving the substrate and the platen relative to one another comprises moving the substrate relative to a stationary platen.

36. The method of claim 34, wherein moving the substrate and the platen relative to one another comprises moving in a first direction and moving in a second direction.

37. The method of claim 36, wherein the first and second directions are opposite directions.

38. The method of claim 37, wherein in moving in opposite directions is repeated at least 2 times.