WO2009086048A1

WO2009086048A1 - Capillary-gap-variance liquid application and removal

Info

Publication number: WO2009086048A1
Application number: PCT/US2008/087586
Authority: WO
Inventors: Vincent R. Rizzo; Roy Rizkovsky; Brian H. Kram; David Chafin; Ryan Reeser; Michael D. Tucker; Kevin D. Marshall; Peter A. Riefenhauser; Lizhen Pang
Original assignee: Ventana Medical Systems, Inc.
Priority date: 2007-12-21
Filing date: 2008-12-19
Publication date: 2009-07-09
Also published as: WO2009085842A1

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, a disclosed apparatus for applying a liquid to a substrate includes 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.

Description

CAPILLARY-GAP- VARIANCE LIQUID APPLICATION AND

REMOVAL

Related Application Data This claims the benefit of U.S. Provisional Patent Application No. 61/015946, filed December 21, 2007 and U.S. Provisional Patent Application No. 61/015951, 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 the actuator exerting a greater force, thus producing a flow of liquid reagent over the substrate. Schembri (EP 089181 IB 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 within a liquid in 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. Release of the tension force causes a liquid to move away from a previously expanded portion of the liquid chamber and impart mixing in the chamber. Similarly, in the device disclosed in PCT Publication No WO 2006/116037 to Erickson et al, two rigid materials are induced to move away from each other in a hinged fashion to induce mixing and redistribution of a liquid within a capillary space. Another hinge-type device that relies on repeatedly separating two rigid materials in an angular fashion to induce mixing within a capillary space is disclosed in U.S. Patent No. 7,285,244 to Gazeau. Hinge-type systems, however, can lead to gradients because of the single type of angular motion that they induce. Furthermore, in the case of the device disclosed by Erickson et al., the clamps used to attach rigid materials to the angular motion inducing mechanism can potentially provide wicking paths that lead to reagents being transported away from the surface to be treated. 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 will be established that draw precious liquids away from a surface of a substrate to be treated.

In one aspect, a disclosed apparatus for applying a liquid to a substrate includes 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 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. In a more particular embodiment, a biological sample to be treated with a liquid is adhered to the lower surface of the substrate between the points where the separators contact the lower surface. In another particular embodiment, the spacers also are located outside a perimeter of the liquid application surface, further minimizing the potential for wicking of liquids away from the capillary space. 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 including a single substrate treatment unit of the aforementioned design. Automation is simplified in the system particularly where forces used to move a liquid within the capillary space are applied directly to the substrate because additional automation to handle a cover or gasket is avoided, as is disposal thereof. Furthermore, if desired, a substrate can easily be automatically loaded into a substrate treatment module without user intervention, for example, to load the substrate into some kind of clip. The disclosed 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.

In yet another aspect, an improved 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 substantially flat liquid application surface and separating the substrate and the liquid application surface at first and second, different sides of the substrate (such as opposite sides) by applying separating forces at the first and second, different sides to a surface of the substrate that is facing the liquid application surface. In one embodiment, the separating force is a lifting force imparted to a lower surface of a horizontally-disposed substrate to move it upwards and away from a horizontal liquid application surface.

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 a top, perspective diagram of an embodiment of a single substrate treatment unit.

FIG. 2 is an exploded, perspective diagram of an embodiment of a single substrate treatment unit shown in FIG. 1.

FIG. 3 is a top view diagram of the treatment platform of an embodiment of a single substrate treatment unit showing a microscope slide positioned over a liquid application surface.

FIG. 4 is a top, perspective diagram of a second embodiment of a single substrate treatment unit.

FIG. 5 is an exploded, perspective diagram of the second embodiment of a single substrate treatment unit shown in FIG. 4. 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 an embodiment 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 separator" refers to one or more separators, such as 2 or more separators, 3 or more separators, or even 4 or more separators.

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 substantially flat liquid application surface, which surface can be made of any material, but is typically constructed from a glass, metal or 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 liquid application surface can include a heater such that the temperature of a liquid in contact with the 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 surface, and multiple such surfaces can be heated or cooled simultaneously or independently.

The disclosed apparatus also includes 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. The spacer can be a liquid that is present in the capillary space, but more typically the spacer can comprise a rail or rails, a tab or tabs, or a pad or pads on or near the liquid application surface or removable spacers adhered to the substrate. In a particular embodiment, protuberances such as adjustable set screws located outside a perimeter of the liquid application surface are used to hold the substrate and the liquid application surface in spaced separation. In another particular embodiment, pads located outside a perimeter of the liquid application surface are utilized to maintain spaced separation. The apparatus also includes 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. The separators can contact the substrate at a point or points of small surface area, or can contact the substrate across a side of a substrate, thereby distributing contact across a larger surface area. In particular embodiments, the separators have knife edges that contact the substrate. The separators can be moved in any manner, for example, they can be mechanically driven, magnetically driven or electrically driven to impart a separating force. The separators can be constructed of any material including metal, glass or plastic, and can be located within the perimeter of the liquid application surface or outside the perimeter of the liquid application surface. In some embodiments, the liquid application surface also includes at least one vacuum port through which the liquid can be removed from the capillary space. In more particular embodiments, a vacuum port is located near an edge of the liquid application surface such that separating the substrate from the liquid application surface at an opposite edge causes the liquid to move toward the vacuum port, thereby increasing the efficiency with which the liquid can be removed from the capillary space through the vacuum port.

In other embodiments, the substrate is held in a depression having at least two sides and a bottom, the bottom of the depression comprising the substantially flat liquid application surface. Optionally, the depression can include one or more reagent delivery wells formed in one or more of the at least two sides of the depression.

In still other embodiments, the substantially flat liquid application surface has an adjacent liquid delivery ramp that is oriented such that liquid deposited onto the ramp will flow from the ramp and onto the liquid application surface. For example, a reagent deposited on the ramp will flow down the ramp and into a capillary space between a substrate and the liquid application surface.

In some embodiments, the at least two separators are independently operable, and in others the separators are operable in a coordinated manner either mechanically or electronically. In more particular embodiments, the apparatus includes at least 3 separators or even at least 4 separators, any number of which can be independently operable or operably-linked in some manner. In other more particular embodiments, operation of the separators is coordinated to occur in some pattern. For example, two separators contacting a substrate on opposite sides of a liquid application surface can operate to alternately and repeatedly widen the capillary space on their respective sides and cause liquid motion back and forth within the capillary space. However, any number of movements and pauses can be combined in a pattern. Larger number of separators can be used to impart more complex motions to the liquid within a capillary space. For example, if 4 separators are disposed near four corners of a 4- sided liquid application surface and are independently operable, a circular motion can be imparted on the liquid by actuating the separators in a pattern of successive actuation around a perimeter of the liquid application surface. Of course, even more complex motions can be imparted using even as few as 3 independently operable separators.

In other particular embodiments, the spacer of the apparatus can comprise a gasket, at least two rails disposed on opposite sides of the liquid application surface, or at least 3 protuberances that contact a surface of the substrate facing the liquid application surface. It should be noted that while a gasket can be used, separation of the substrate from the liquid application surface will not cause compression of the gasket as in prior systems. For example, in the particular case of a horizontally disposed liquid application surface with a substrate resting horizontally on a gasket above the liquid application surface, the separators will lift the substrate off of the gasket. In other particular embodiments, where protuberances are used as the spacer, the protuberances can be adjustable, either manually or automatically to vary the separation between the substrate and the liquid application surface, and thereby vary the volume of the capillary space. Set screws are one example of adjustable protuberances that can be used in the disclosed apparatus. Alternatively, protuberant spacers such as pads can be fixed and the separation between the substrate and the liquid application surface can be adjusted by movement of the liquid application surface, for example, by spring loaded screws as illustrated in FIG. 5. Although described as such immediately above and in the particular working embodiments that follow, having the liquid application station disposed horizontally and the substrate disposed horizontally above the liquid application station is only one possible configuration for the disclosed apparatus. For example, it is possible to have the substrate on the bottom and the liquid application surface above. In this instance, the separators would move the liquid application surface up and away from the substrate by pushing down onto a surface of the substrate facing the liquid application surface. Alternatively, both the substrate and the liquid application station could be disposed vertically, or any angle in between horizontal and vertical. Furthermore either one of the substrate and the liquid application surface can be held stationary and the other moved, or both the substrate and the liquid application surface could move in response to a separating force imparted by the separators.

Disclosed embodiments of the apparatus can further include a liquid applicator configured to deliver the liquid to the capillary space. The liquid applicator can be, for example, one or more of an aperture through the liquid application surface, a stationary or moveable nozzle, and a robotic dispenser. 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. The single substrate treatment modules each include a single substrate treatment unit. The single substrate treatment units include 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 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. 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 module 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 can include 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 once that portion has moved past the depression(s).

It is not only 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 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 substantially flat liquid application surface, separating the substrate and the liquid application surface at a first side by applying a separating force on the first side to a surface of the substrate that is facing the liquid application surface and moving the substrate away from the liquid application surface at the first side; and separating the substrate and the liquid application surface at a second, different side (such as an opposite side) by applying a separating force on the second side to the surface of the substrate that is facing the liquid application surface and moving the substrate away from the liquid application surface at the second side. In a particular embodiment, separating on the first side and separating on the second side comprise lifting the first side and lifting the second side by applying lifting forces to a bottom surface of the substrate at the first and second sides to push the first side and the second side upward from a stationary, horizontal liquid application surface. In other particular embodiments, separating on the first side and separating on the second side are alternated. In still other particular embodiments, separating on the first side and separating on the second side are alternately repeated. In yet other particular embodiments, the method further includes separating the substrate and the liquid application surface at one or more additional sides, which can, for example, be done in a pattern such as around the perimeter of the substrate as was discussed above.

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, the substantially flat liquid application surface 12 includes a vacuum port 13, and the spacer comprises 4 set-screws 14 near but separated from and outside the four corners of the liquid application surface 12. Separators 16, 17 are made from a thin metal material and shaped to contact a substrate placed onto the spacer screws 14 and are driven upward by stepper motor 38 to lift the respective ends of a substrate that they contact. Liquid application surface 12 is bounded on two sides by sloped walls 18 such that the liquid application surface is within a depression in treatment platform 26. A plurality of liquid delivery wells 20 formed in the walls 18 is shown in this embodiment.. In some embodiments of a system or single substrate treatment module incorporating a disclosed single substrate treatment unit, a robotic pipettor is used to deliver liquids to the larger well, and dedicated nozzles are used to dispense the same or different bulk reagents into the three smaller wells. Substrate stops 22 and 24 function to hold a substrate in position longitudinally over the liquid application surface 12, while walls 18 serve to hold a substrate in position laterally over the liquid application surface. The separation between the liquid application surface and a facing surface of a substrate can be adjusted using the set screws 14.

FIG. 2 shows an exploded view of an embodiment of a single substrate treatment unit 10. In this view, a mechanism is shown that can be used to move either of separators 16 and 17 upward such that a substrate is lifted off of set-screws 14 on one end or the other of the opposite sides of the substantially flat liquid application surface 12. Separators 16 and 17 are attached to opposite ends of rocker arm assembly 30, which assembly rotates about axle 32. Axle 32 is held in bearings 33 attached to opposite side supports 27 and 29, which side supports are connected to treatment platform 26 and lateral support 28. Connector 34 that is mounted to rocker arm assembly 30 below axle 32 has a threaded head portion into which threaded rod 36 is inserted. Stepper motor 38 rotates threaded rod 36, and as threaded rod 36 is rotated connector 34 is either drawn toward the stepper motor 38 or pushed away from the stepper motor. Since connector 34 is mounted to rocker arm assembly 30 off-axis, movement of the connector 34 toward or away from stepper motor 38 causes rocker arm assembly 30 to rotate around axle 32. As rocker arm assembly 30 is rotated, separators 16 and 17 are each either pushed upward above the treatment platform 26 or pulled down below (and through a hole in the platform in the case of 16). Since separators 16 and 17 are attached to the same rotating assembly one will go upward as the other goes down.

FIG. 3 shows how a substrate 40 (in this case a microscope slide) rests on set- screws 14 within the depression formed in the treatment platform 26. Substrate stops 22 and 24 limit longitudinal movement while the depression limits lateral movement. In operation (with reference to FIGS. 1-2), a liquid delivered to a liquid delivery well 20 will be drawn by capillary forces under the slide and will spread out into the capillary space between the slide and the substantially flat liquid application surface 12. Mixing and/or redistribution of the liquid within the capillary space is induced by raising the slide off of the set screws 14 with the separators 16 and 17. Opposite ends of the slide can be alternately raised one or more times, but more typically at least several times, to cause mixing and/or redistribution of the liquid. A liquid between the slide and the liquid application surface will move away from the raised end of the slide. Thus, in some embodiments, removal of a liquid from the capillary space can be accomplished by a combination of vacuum being applied at vacuum port 13 simultaneous with lifting the opposite end of the slide to cause the liquid to move toward the vacuum port. In practice, the separators can be configured to provide minimal lift of an end of a slide such that the liquid moves only a small distance toward the opposite end of the capillary space, or configured to provide a maximal lift such that the liquid moves substantially toward the opposite end of the capillary space. Any combination of minimal and maximal lifts at one or both ends of the length of the slide can be employed.

FIG. 4 shows a perspective view of another embodiment of a single substrate treatment unit 100 that does not include a depression into which a substrate is placed. Also, in this embodiment, the spacing between a substrate and the substantially flat liquid application surface 12 is adjusted by moving the application surface relative to a fixed position for the substrate rather than moving the substrate relative to a fixed position for the application surface. The embodiment of FIG. 4 includes liquid delivery ramp 50. Liquid delivery ramp 50 is a tilted surface adjoining the application surface 12 that is angled downward toward the application surface. Liquids dispensed onto liquid delivery ramp 50 will thus flow down onto the application surface 12 and into a capillary space between the application surface and a substrate held in spaced relationship with the application surface. In this particular embodiment, substrate stops 22 and 24 again serve to limit longitudinal movement of a substrate, while posts 52 serve to limit lateral movement of a substrate. Within the longitudinal and lateral bounds established by the stops and posts, a substrate can be placed onto pads 54 that establish a fixed position for the substrate relative to treatment platform 26. The spacing between substantially flat liquid application surface 12 and a substrate can be adjusted with height adjustment screws 56 against the heads of which the application surface is urged by liquid application surface support springs 57 that are shown in FIG. 5. In this embodiment, separators 16 and 17 are alternately urged upward to lift opposite ends of a substrate off of the respective pads 54 on a particular end to cause liquid to move within the capillary space between the application surface and the substrate. In this particular example, the mechanism used to actuate the separators includes stepper motor 38, sliding block 58 that is moved back and forth within channel 55 by the stepper motor and flag 60 against which the sliding block rests. Sliding block 58 can be made of any material, but a particularly useful material is DELRIN which is easily machined to include a threaded hole into which a threaded rod attached to a stepper motor can be introduced. In operation, stepper motor 38 turns a threaded rod in one direction and drives sliding block 58 into flag 60, thereby urging separator 16 upward and separator 17 downward. When the direction of stepper motor is reversed and the sliding block 58 is drawn back toward the stepper motor 38. A return spring 65 (see, FIG. 5) urges separator 17 upward and separator 16 downward as the sliding block is drawn back toward the stepper motor. Sensor 70 is used to detect a condition when neither separator 16 nor separator 17 is lifting a substrate off of pads 54, thus establishing a "zero" position.

FIG. 5 shows an exploded diagram of the embodiment of a single substrate treatment unit 10 shown in FIG. 4. In this view, separators 16 and 17 are shown as components of rocker arm halves 62 and 63. Rocker arm halves 62 and 63 rotate around shoulder bolt 61. The angle between the rocker arm halves can be adjusted with rocker arm set screw 64. Sensor set screw 72 is adjustable such that it contacts sensor 70 at the proper "zero" position. Also illustrated in FIG. 5 is nose portion 59 of sliding block 58 that contacts flag 60 and is rounded such that the sliding block can move in a single plane as the flag is pushed upward. Threaded rod 36 is shown as a shaft extending from stepper motor 38. Further illustrated in FIG. 5 are liquid application surface support springs 57, and vacuum line 80 leading from vacuum hole 13 of substantially flat liquid application surface 12.

An advantage of the mechanism for moving a substrate away from a liquid application surface shown in FIGS. 4 and 5 is that there is less storage of force in the drive mechanism during the movement process. The force required to separate a substrate from an application surface when a liquid is present in a capillary space between them is greatest at first when the surface area across which the substrate and application surface are held together by the liquid in the capillary gap is greatest. If there is a tendency in the drive mechanism to store energy, once the capillary forces are initially overcome, extra energy stored in the drive mechanism will suddenly be released as the force needed to separate decreases with decreasing surface area of contact and the substrate will "pop" away from the liquid application surface. 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 predetermined 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 substantially flat liquid application surface; 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 different 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.

2. The apparatus of claim 1, wherein the liquid application surface includes at least one vacuum port through which the liquid can be removed from the capillary space.

3. The apparatus of claim 1 , wherein the substrate is held in a depression having at least two sides and a bottom, the bottom of the depression comprising the substantially flat liquid application surface.

4. The apparatus of claim 3, wherein the depression includes a reagent delivery well formed in one or more of the at least two sides of the depression.

5. The apparatus of claim 1 , wherein the at least two separators are independently operable.

6. The apparatus of claim 1, comprising at least 3 separators.

7. The apparatus of claim 6, wherein the at least 3 separators are independently operable.

8. The apparatus of claim 1, comprising at least 4 separators.

9. The apparatus of claim 8, wherein the at least 4 separators are independently operable.

10. The apparatus of claim 8, wherein the at least 4 separators are disposed such that 2 separators are disposed on one side of the liquid application surface and 2 separators are disposed on the opposite side of the liquid application station.

11. The apparatus of claim 10, wherein the at least 4 separators are disposed near four corners of a 4-sided liquid application surface.

12. The apparatus of claim 1 1 , wherein the at least 4 separators are independently operable and are configured to move the substrate away from the liquid application surface in a pattern.

13. The apparatus of claim 12, wherein the pattern comprises successive actuation of the separators around a perimeter of the liquid application surface.

14. The apparatus of claim 1, wherein the separators are located outside a perimeter of the liquid application surface.

15. The apparatus of claim 1 , wherein the spacer comprises a gasket, at least two rails disposed on opposite sides of the liquid application surface, or at least 3 protuberances that contact a surface of the substrate facing the liquid application surface.

16. The apparatus of claim 1, at least one of the at least 3 protuberances are adjustable such that the spaced separation between the substrate and the liquid application surface can be varied.

17. The apparatus of claim 1, wherein the liquid application surface and the substrate are substantially horizontal.

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

19. The apparatus of claim 1 , wherein the substrate is moved by the separators and the liquid application surface is stationary.

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

21. The apparatus of claim 20, wherein the liquid applicator comprises one or more of an aperture through the liquid application surface, a stationary or moveable nozzle, and a robotic dispenser.

22. An automated system for treating a plurality of substantially flat substrates with a liquid, comprising: a plurality of single substrate treatment modules; the single substrate treatment modules each comprising a single substrate treatment unit; the 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, 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.

23. The system of claim 22, further including one or more of a substrate transporter, a substrate drying unit, a substrate holding cassette, a machine-readable code reader and a reagent transporter.

24. The system of claim 22, wherein the liquid delivery system comprises a dedicated robotic dispenser in each of the plurality of single substrate treatment modules.

25. The system of claim 22, wherein the liquid delivery system comprises a multi-well reagent pack and a reagent pack delivery system.

26. The system of claim 25, 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.

27. The system of claim 22, wherein one or both of the substrate and a reagent container are labeled with a machine-readable code.

28. The system of claim 22, 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.

29. The system of claim 22, further comprising one or more additional single substrate treatment modules; the additional substrate treatment modules comprising an alternate single substrate treatment unit; the alternate 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.

30. The system of claim 22, wherein single substrate treatment units are interchangeable between the plurality of single substrate treatment modules.

31. The system of claim 29, wherein the single substrate treatment unit and the alternate single substrate treatment unit are interchangeable between the single substrate treatment module and the additional single substrate treatment module.

33. A method for applying a liquid to a substantially flat substrate, comprising: introducing the liquid into a capillary space between the substrate and a substantially flat liquid application surface; separating the substrate and the liquid application surface at a first side by applying a separating force on the first side to a surface of the substrate that is facing the liquid application surface and moving the substrate away from the liquid application surface at the first side; and separating the substrate and the liquid application surface at a second, different side by applying a separating force on the second side to the surface of the substrate that is facing the liquid application surface and moving the substrate away from the liquid application surface at the second side.

34. The method of claim 33, wherein separating on the first side and separating on the second side comprise lifting the first side and lifting the second side by applying lifting forces to a bottom surface of the substrate at the first and second sides to push the first side and the second side upward from a stationary, horizontal liquid application surface.

35. The method of claim 33, wherein separating on the first side and separating on the second side are alternated.

36. The method of claim 33, wherein separating on the first side and separating on the second side are alternately repeated.

37. The method of claim 33, further comprising separating the substrate and the liquid application surface at one or more additional sides.

38. The method of claim 37, wherein separating the substrate and liquid application surface at the first side, at the second side and at one or more additional sides is done in a pattern.

39. The method of claim 38, wherein separating in a pattern comprises sequentially separating the substrate and the liquid application surface around a perimeter of the substrate that defines the sides of the substrate.