WO2010074915A2

WO2010074915A2 - Evaporation mitigation for slide staining apparatus

Info

Publication number: WO2010074915A2
Application number: PCT/US2009/066568
Authority: WO
Inventors: Brian H. Kram
Original assignee: Ventana Medical Systems, Inc.
Priority date: 2008-12-23
Filing date: 2009-12-03
Publication date: 2010-07-01
Also published as: WO2010074915A3

Abstract

A device for mitigating evaporation of treatment liquids from a sample on a substrate can include a solvent reservoir and a conduit. A slide with a sample on a surface thereof can be placed into a device, creating a capillary gap between the slide surface and a surface of the device. Treatment fluids can be dispensed and drawn into the capillary gap, such as by capillary action. The slide and/or the reservoir can be moved towards the conduit, such that the fluid in the capillary gap contacts a fluid meniscus formed in the conduit. In this manner, fluid lost to evaporation from the capillary gap can be replenished by fluid in the conduit and reservoir, via capillary action. The slide and/or reservoir can then be moved again to break contact with the conduit, and the fluid within the capillary gap can then be mixed, such as by rocking the slide.

Description

EVAPORATION MITIGATION FOR SLIDE STAINING APPARATUS

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to and the benefit of U.S. Provisional Patent Application No. 61/140,259 filed December 23, 2008, and is incorporated herein in its entirety.

FIELD

The present disclosure concerns embodiments of an apparatus for staining a sample on the surface of a support member, and methods for its use. Specifically, the disclosure concerns a tissue staining apparatus for mitigating fluid evaporation from a microscope slide.

BACKGROUND A sample, such as a human or animal tissue sample, can be fixed onto a substrate, such as a glass microscope slide. Analysis of the sample may require that the sample be treated and/or stained, such as by applying one or more liquids to the sample on the slide.

Treatment reagents can be quite expensive, and thus efforts have been made to minimize the volumes of such liquids required for treating a sample. One such method for applying fluids to a biological sample is disclosed in U.S. Application No. 11/187,183, which is incorporated herein by reference.

In conventional methods, a small amount of a liquid, such as less than about 100 μL, and often less than 50 μL, is applied to a sample on a microscope slide. However, such treatment liquids often evaporate, especially when sample processing requires that heat be applied to the sample in a thermal cycle. Reagent evaporation can decrease sample quality due to drying, and/or cause reagent constituent precipitation, loss of concentration control, loss of kinetic control, non-uniform coverage of treatment liquids, and/or sample damage. Various methods have been employed to prevent or mitigate evaporation of such treatment liquid. One such method of preventing or reducing treatment liquid evaporation involves applying a liquid coverslip to the sample, thereby trapping the treatment liquid in between the sample and the liquid coverslip. One example of liquid coverslips for use with the NexES® systems (Ventana Medical Systems, Inc., Tucson, AZ), use oil to provide a fluid seal over an aqueous chemistry layer.

Another method involves creating a mechanical seal around the sample and treatment liquid, such as by creating a chamber sealed to the slide or to a base plate by rubber gaskets or o-rings. Such mechanical seals can create very small microchambers that are air and/or liquid impermeable and completely enclose the sample and treatment liquids on the slide.

Using a humidification chamber is yet another conventional method of substantially eliminating or reducing evaporation. In this method, the entire slide and sample are placed within a chamber. The chamber humidity can be controlled to mitigate evaporation. Finally, an open opposed plate method utilizes a second, non-permeable, surface that is provided parallel to and opposing the surface of the microscope slide. The opposing surface covers most of the sample and treatment liquid, but portions of the liquid may be exposed to the environment (e.g., at the sides and ends of the opposing surface), and thus subject to evaporation. Because some of the treatment liquid remains exposed, the open opposed plate method often requires replenishment of expensive reagent fluids, especially during processing where the sample is heated (e.g., high temperature antigen retrieval).

The open opposed plate method provides advantages over devices such as a humidification chamber or mechanically sealed chamber, by allowing direct access to the sample and slide. Thus, the slide can be easily moved to and from the processing area, and reagents and other treatment liquids can easily be added to the sample. Open opposed plate devices and methods also are simpler and less expensive to manufacture than humidification and mechanically sealed chambers. Lower complexity also lends greater reliability. However, as mentioned, the open opposed plate method often requires replenishing expensive reagent fluids and/or other evaporation mitigation methodologies. Therefore, a need remains for improved methods and devices for mitigating evaporation of treatment liquids from a substrate.

SUMMARY

The present method and apparatus provide improved evaporation mitigation by semi-continuous solvent replenishment in a manner conducive to automation. Exemplary devices can include micro fluidic devices and features.

One embodiment of a device according to the present disclosure comprises a slide support designed to accommodate a slide having a sample on a first surface thereof. An opposed surface can be arranged to be opposed to and spaced apart from the slide surface to form a capillary gap. A fluid reservoir periodically replenishes fluid in the capillary gap. A conduit can provide temporary fluid communication between fluid in the reservoir and fluid in the capillary gap. The device can comprise a sensor to detect fluid levels. For example, certain disclosed embodiments include a reservoir sensor to provide feedback as to actual fluid volume levels in the reservoir and/or a capillary gap sensor to provide feedback regarding evaporation and/or fluid levels within the capillary gap.

The slide support can allow movement of the slide, such as movement between a first position in which a first liquid within the capillary gap is displaced (e.g., laterally displaced) from the conduit, and a second position in which the first liquid within the capillary gap is brought into contact with a second liquid within the conduit. In some embodiments, the slide is movable from the first position to the second position by translation of the slide in a horizontal plane. Additionally or alternatively, the slide can be movable in a vertical and/or orthogonal plane. In some embodiments, the slide can be moveable in the horizontal plane in a second direction, wherein the second direction is at an angle to a first direction of movement between the first and second positions of the slide. For example, the slide can be moveable between a first position and a second position by translating the slide in the x direction. The slide can also be movable in the y direction (e.g., in a direction at an angle to the first direction of movement) to, for example, promote fluid mixing in the capillary gap.

In some embodiments, the device can allow movement of the reservoir, such as movement between a first position in which a first liquid in the reservoir is displaced from the conduit, and a second position in which the first liquid within the reservoir is brought into contact with the conduit and/or a second liquid within the conduit. In some embodiments, the reservoir is movable from the first position to the second position by translation of the reservoir in a horizontal plane. Additionally or alternatively, the reservoir can be movable in a vertical and/or orthogonal plane.

In some embodiments, both the reservoir and the slide are movable. In some embodiments, only one of either the slide or the reservoir is movable. In some embodiments where only the reservoir is movable, the slide can be positioned such that the first liquid in the capillary gap contacts the liquid in the conduit. Some disclosed embodiments of a device for mitigating evaporation comprise a rocking mechanism configured to mix the fluid in the capillary gap. In some embodiments, the rocking mechanism comprises one or more pins configured to raise and lower alternating sides of the slide.

The reservoir can be tilted, or oriented at an angle with respect to the slide and/or with respect to the opposing surface. Additionally or alternatively, some disclosed embodiments of a device comprise a reagent dispense area in fluid communication with the capillary gap. The reagent dispense area, for example, can allow addition of a fluid, such as a reagent, to the capillary gap, without requiring direct access to or exposure of the capillary gap. The reagent dispense area can be provided, for example, on the opposite side of the slide from the area of the capillary gap that contacts the conduit.

Devices according to the present disclosure can at least partially rely on fluid tension and capillary action to control fluid movement, rather than actuating mechanisms. For example, the fluid tension of the first liquid in the capillary gap can be greater than the fluid tension of the second liquid in the reservoir. This can prevent fluid from flowing from the capillary gap back into the conduit and/or reservoir, without requiring valves or other such features. Similarly, in some embodiments, fluid tension prevents the second liquid from escaping the conduit until the second liquid contacts the first liquid in the capillary gap.

The distance between the slide surface and the second surface, and hence the volume of the capillary gap, can be determined and maintained in many different ways. In one embodiment, at least two parallel rails are provided between the slide surface and the second surface, thereby defining the height or thickness of the capillary gap.

Some disclosed embodiments of a device according to the present disclosure comprise a slide having a sample on a surface thereof and a curved surface opposed to the slide surface. The curved surface can include a reservoir, a conduit in fluid communication with the reservoir, and a mixing trough. The slide can be movable relative to the curved surface such that at least a portion of a liquid between the slide surface and the curved surface is moved into and out of the mixing trough as the slide moves.

Some disclosed embodiments of a device that include a curved surface comprise at least two mixing troughs provided in the curved surface. In some embodiments, the mixing trough can be located in the curved surface at a location remote from the reservoir. Embodiments of a device can comprise a sensor to detect fluid levels in the reservoir and/or in the capillary gap.

Various embodiments of a method for mitigating evaporation of a treatment liquid from a microscope slide are disclosed. One such embodiment comprises providing a thin sample on a first surface, maintaining a second surface opposed to and spaced by a first distance from the first surface, thereby providing a capillary gap between the first and second surfaces, and providing a first liquid within the capillary gap. A second liquid can be dispensed into a reservoir in fluid communication with a conduit, such that the second liquid fills the conduit via capillary action. The first liquid in the capillary gap can be brought into contact with the second liquid in the conduit. The second liquid then flows from the conduit into the capillary gap to fill any voids formed in the capillary gap due to evaporative loss of the first liquid. The method can include breaking the contact between the first liquid in the capillary gap and the second liquid in the conduit and mixing the second liquid with the first liquid.

In some embodiments according to the present disclosure, mixing the second liquid with the first liquid comprises rocking the slide, such rocking the slide using a rocking mechanism.

Some embodiments of a method comprise contacting the first liquid in the capillary gap with the second liquid in the conduit and breaking the contact periodically. For example, a slide can be moved by an actuating device such that fluid in the capillary gap contacts fluid in the conduit, and then moved by an actuating device in an opposite direction to break the contact. After a set period of time, or when a user notices voids forming in the capillary gap, the slide again can be actuated so that fluid in the capillary gap contacts fluid in the conduit. The contact can be broken after a predetermined period of time and/or after a period of time determined by a user. Furthermore, contacting and breaking contact can be alternately repeated according to an automated time schedule, or manually, as determined necessary by a user.

In some embodiments, the second, opposing, surface is substantially parallel to the first surface. In some embodiments, the opposing surface is a curved surface, such as a curved plate. In these embodiments, mixing the second liquid with the first liquid can comprise rocking the slide back and forth over the curved surface. Embodiments including providing a curved surface can also include providing a curved surface having a mixing trough.

The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating one embodiment of a device for mitigating evaporation according to the present disclosure. FIG. 2 is a top plan view of one embodiment of a device for mitigating evaporation, with the slide in a first position. FIG. 3 is a top plan view of the device of FIG. 2, with the slide in a second position.

FIG. 4 is a perspective view of the device of FIG. 2. FIG. 5 is a perspective view of one embodiment of a rocker mechanism according to the present disclosure.

FIG. 6 is a cross-sectional view illustrating another embodiment of a device for mitigating evaporation.

FIG. 7 is a perspective view of the device of FIG. 6. FIG. 8 is a block diagram illustrating one embodiment of a method for mitigating evaporation of treatment liquids from a surface.

FIG. 9 is a cross-sectional view taken along line 9-9 in FIG. 2.

DETAILED DESCRIPTION

As used in this application and in the claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

Additionally, the term "includes" means "comprises." Further, the term "coupled" means coupled in a manner effective to achieve a stated or understood goal, and includes, but may not be limited to, physically, mechanically, electrically, or optically coupled or linked and does not exclude the presence of intermediate elements between the coupled items.

Although the operations of embodiments of the disclosed method may be described in a particular, sequential order for convenient presentation, it should be understood that this description encompasses rearrangements, unless a particular order is required in context by specific language. For example, operations described sequentially may in some cases be rearranged or performed concurrently. Moreover, the attached figures may not show the various ways in which the disclosed system, method, and apparatus can be used in conjunction with other systems, methods, and apparatus. Additionally, the description sometimes uses terms like "provide" to describe the disclosed method. These terms may be high- level abstractions of the actual operations that can be performed. The actual operations that correspond to these terms can vary depending on the particular implementation and are discernible by a person of ordinary skill in the art.

FIG. 1 is a schematic diagram illustrating one embodiment of a device for mitigating evaporation according to the present disclosure. A tissue staining apparatus 100 can include a surface 102 opposed to the surface of slide 104. The slide 104 includes a sample, such as a biological tissue sample. Slide 104, and hence the sample, is positioned such that the sample faces the opposing surface 102. Furthermore, slide 104 is positioned relative to surface 102 to form a small gap, such as a capillary gap, between the sample bearing surface of slide 104 and the opposing surface 102. This small gap can contain one or more liquids to be added to and/or reacted with the sample on the surface of slide 104.

Surface 102 can include a reservoir, such as a micro fluidic reservoir 106 in fluid communication with a micro fluidic conduit or slit 108. A dispenser 110 can be used to dispense one or more treatment liquids onto the opposing surface 102 and/or into the reservoir 106. For example, dispenser 110 can dispense a volume of solvent or fluid reagent into reservoir 106. Dispenser 110 can be, for example, a micropipette manipulated by a user, and/or an automated pipetting mechanism.

In some embodiments, both the reservoir 106 and the conduit 108 fill spontaneously due to capillary action when a liquid is dispensed into the reservoir 106.

In some embodiments, the slide 104 can be reciprocated back and forth in the directions of arrow 112. For example, the slide 104 can be reciprocated in the directions of arrow 112 such that it moves from a first position to a second position. In a first position, the slide 104 can be positioned such that it (and therefore the capillary gap between it and the opposed surface 102) is displaced (e.g., displaced laterally) away from the conduit 108. In a second position, the slide 104 can contact the conduit 108. Specifically, the fluid in the capillary gap can be made to contact the fluid contained in the conduit 108. When slide 104 is in the second position, the sample and liquids in the capillary gap are in fluid communication with the fluids in the conduit 108 and reservoir 106 through the conduit 108. If fluid evaporates, a small void may form in the liquid contained in the capillary gap between the slide 104 and the opposed surface 102. When the slide 104 is brought into contact with the conduit 108, fluid contained in the conduit 108, such as a replenishing solvent liquid, can flow from the reservoir 106, into the conduit 108, and into the capillary gap to replace the evaporated fluid. In some embodiments, the flow can automatically cease once the capillary gap is refilled, due to satisfaction of capillary gap fluid tension.

Contact between the slide 104 and the conduit 108 can then be broken by moving the slide 104, such as along arrow 112 back to the first position, such that it is again displaced from the conduit 108. Once contact has been broken, the slide 104 can be manipulated to effect fluid mixing in the capillary gap. In one embodiment, the slide 104 can be reciprocated back and forth in the directions of arrow 114 to promote fluid mixing in the capillary gap. In some embodiments, the slide 104 can additionally or alternatively be rocked or tilted, such as being rocked or tilted along the direction of arrow 114.

In some embodiments, the slide 104 can remain in contact with the conduit 108. In these embodiments, the reservoir 106 can be movable, such as by reciprocating the reservoir 106 in the direction of arrows 112 and/or 114 away from and towards the conduit 108. For example, the reservoir 106 can be reciprocated in the directions of arrow 112 such that it moves from a first position to a second position. In a first position, the reservoir 106 can be positioned such that it is displaced (e.g., displaced laterally) away from the conduit 108. In a second position, the reservoir 106 can contact the conduit 108. Specifically, the fluid in the reservoir 106 can be made to contact the fluid contained in the conduit 108. When reservoir 106 is in the second position, the sample and liquids in the capillary gap can be in fluid communication with the fluids in the conduit 108 and reservoir 106 through the conduit 108.

Contact between the reservoir 106 and the conduit 108 can then be broken by moving the reservoir 106, such as along arrow 112 back to the first position, such that it is again displaced from the conduit 108. The directions indicated by arrows 112 and 114 are intended to be exemplary and not limiting. The reservoir 106 and/or the slide 104 can be movable in any direction, to achieve displacement between the slide 104 and the conduit 108 and/or displacement between the reservoir 106 and the conduit 108.

In the embodiment shown in FIG. 1, the directions indicated by arrow 114 are substantially perpendicular to the directions indicated by arrow 112. However, in other embodiments, the direction of manipulation for contacting the conduit 108 (e.g., the directions indicated by arrow 112) need not be perpendicular to the direction of manipulation for mixing fluid in the capillary gap (e.g., the directions indicated by arrow 114). For example, in some embodiments, the directions of manipulation can be substantially the same. For example, manipulating the slide 104 along arrow 112 can also promote mixing in some embodiments. In some embodiments, the range of motion for fluid mixing can be less than the range of motion for contacting the conduit 108. In this manner, there is no contact between the slide 104 and the conduit 108 during mixing. In other embodiments, the range of motion for fluid mixing can be greater than the range of motion for contacting the conduit 108. For example, translation of the slide 104 along arrow 112 to contact the conduit 108 can be a relatively small movement, while movement of the slide 104 for mixing the fluid in the capillary gap can be a comparatively large movement. In some embodiments, the directions of manipulation can be at an angle to one another, such as at an angle of between zero and ninety degrees, or at an angle of between ninety and 180 degrees.

In some embodiments, the direction of manipulations as indicated by arrows 112 and 114 do not occur in the same plane. For example, manipulating the slide 104 along arrow 112 can include reciprocating the slide back and forth within a horizontal x-y plane, and manipulating the slide 104 along arrow 114 can include moving the slide 104 in a z plane orthogonal to the x-y plane. Additionally, manipulating the slide 104 for either mixing or contacting the conduit 108 can occur in more than one plane at a time, or more than one plane at separate times.

The device 100 can define a fixed distance between the surface of slide 104 and the opposing surface 102, thereby defining a fixed capillary gap volume therebetween. The fixed capillary gap volume can prevent overfilling on the slide surface. Control of the fluid tension in the reservoir 106 and conduit 108 can prevent excess liquid from wicking or dripping out of the capillary gap boundaries. Appropriate control of fluid tension can also ensure that fluid flow is unidirectional. Thus, the device 100 can essentially perform the function of valves without actually including valves in the device. Such valving can be performed via the controlled fluid tensions and by breaking and providing contact between the capillary gap and the conduit 108 and/or by breaking and providing contact between the reservoir 106 and the conduit 108.

Appropriate fluid flow can be controlled by fluid tension relationships between the fluid in the reservoir 106, conduit 108, and the capillary gap under slide 104. Such fluid tension relationships can be maintained by controlling the fluid chemistry used, the surface interactions between the fluid and the reservoir 106, conduit 108, or capillary gap, and/or by the dimensions and geometries of the reservoir 106, conduit 108, and capillary gap. For example, different volumes of processing fluids can be used in various methods of treating a sample on a slide.

The height and the surface area of the capillary gap define the volume of liquid that the capillary gap will hold. Thus, processing that requires a larger volume of treating liquids can be accommodated with a capillary gap with a greater height (e.g., a greater distance between the slide surface and the opposing surface 102). For example, for processing that requires a lesser volume of treating fluid, such as 40 μL of treating fluid, a smaller capillary gap height can be provided than when the processing requires a greater volume of treating fluid, such as 100 μL of treating liquid. In some embodiments, features such as rails can define the height of the capillary gap, and thus a device designed for, for example, 40 μL processing methods may include smaller rails than would be included in a device designed for, for example, 100 μL processing methods.

In some embodiments, the height of the capillary gap can be from about 0.001 inches to about 0.010 inches. In some embodiments, the height of the capillary gap can be from about 0.002 inches to about 0.004 inches. In one embodiment, the height of the capillary gap can be about 0.003 inches. Capillary gaps of different heights can have different fluid tensions. For example, a larger capillary gap (e.g., a capillary gap with a greater height) can have less fluid tension than a smaller capillary gap. In some embodiments, the conduit 108 can have less fluid tension than the capillary gap, such as by having a height greater than the height or thickness of the capillary gap. In some embodiments, the height of the conduit 108 can be about 0.006 inches. In some embodiments, the reservoir 106 can have less fluid tension than the capillary gap and/or the conduit 108. For example, at least a portion of the reservoir 106 can have a height greater than the height of the conduit 108. In some embodiments, the reservoir 106 can have a range of heights, where the reservoir 106 decreases in height near the conduit 108. For example, the reservoir 106 can decrease in height along its length, such as decreasing from a height of about 0.020 inches near the end of the reservoir 106 farthest from the conduit 108, to a height of about 0.010 inches near the end of the reservoir 106 closest to the conduit 108.

In some embodiments, the fluid in the capillary gap between the surface of slide 104 and the opposing surface 102 exhibits a higher fluid tension than the fluid in the reservoir 106. In this manner, any overfill or extra liquid dispensed, can spill out of the reservoir 106, rather than flowing into the capillary gap.

The conduit 108 can be self- filling and self-limiting in some embodiments. For example, when in contact with the reservoir 106, the conduit 108 can fill automatically with fluid from the reservoir 106 without requiring a separate valve or filling mechanism. Fluid tension within the conduit 108 can prevent the fluid from escaping the conduit 108.

The reservoir 106 can also include defined surfaces that exhibit capillary wicking of fluid. In this manner, the reservoir 106 can tolerate a dynamic range of fill volumes. For example, in some embodiments, the reservoir 106 can be tilted with respect to the plane of the slide 104 and/or with respect to the plane of the opposed surface 102. The reservoir can be designed to include a height gradient. Thus, if the reservoir 106 is not completely full, the design of the reservoir 106 can ensure that the present fluid remains in one area of the reservoir 106. For example, the design of the reservoir 106 can ensure that the present fluid remains in an area of the reservoir closest to the conduit 108 so that the fluid within the reservoir 106 is in fluid communication with the conduit 108 when the reservoir 106 is positioned to contact the conduit 108. In some embodiments, any voids present in the reservoir 106 are kept in an area of the reservoir 106 farthest away from the conduit 108 by virtue of the capillary action within the reservoir 106. In some embodiments, excess fluid or reservoir over-fill can be shunted away to prevent fluid communication with the fluid contained in the conduit 108 and/or capillary gap. This can help to maintain desired fluid tension relationships between the reservoir 106, conduit 108, and capillary gap under the slide 104. For example, in some embodiments, the lowest fluid tension in the reservoir 106 can be greater than the fluid tension at the boundary of the capillary gap in order to prevent excess fluid from flowing out at the capillary gap boundaries.

Some embodiments include a filling or dispensing area for filling the reservoir 106. For example, a solvent dispense area can be provided in fluid communication with the reservoir 106. A user can dispense fluid into the solvent dispense area using a pipette, and/or an automated device can dispense fluid into the solvent dispense area. The reservoir 106 can fill from this dispensed fluid, and excess fluid can remain in the solvent dispense area. In some embodiments, dispensing volume need not be precisely controlled. In some embodiments, excess fluid can flow down the length of the reservoir 106, away from the slide 104. The frequency of bringing the slide 104 and/or the reservoir 106 into contact with the conduit 108 can be controlled automatically and/or manually. In some embodiments, the slide 104 and/or reservoir 106 contacts the conduit 108 on a periodic basis, such as between about 1 and about 40 times per minute. In some embodiments, the slide 104 and/or reservoir 106 can be moved continuously between contacting and not contacting the conduit 108. In some embodiments, the slide 104 and/or reservoir 106 can remain away from the conduit for a period of time before it is brought into contact with the conduit 108 again. In some embodiments, this can allow time for mixing of the fluid in the capillary gap.

In some embodiments, the slide 104 and/or reservoir 106 is brought into contact with the conduit 108 as frequently as deemed necessary by a user and/or monitoring device. In some embodiments, the slide 104 and/or reservoir 106 can be both periodically brought into contact with the conduit 108 and additionally brought to contact the conduit 108 when voids form in the capillary gap between periodic contacting. The frequency of contacting can be increased or decreased depending on the details of a particular reaction or processing method. For example, in methods involving high temperature thermal cycling, contacting frequency may be higher than the contacting frequency for methods that take place at room temperature.

Similarly, the frequency of moving (e.g., rocking, rolling, or reciprocating) the slide 104 for mixing can be controlled automatically and/or manually. In embodiments where mixing is accomplished by rocking the slide 104, a cycle can be defined as lifting a first end of the slide 104, lowering the first end, lifting a second end of the slide 104, and lowering the second end. Rocking can be performed at, for example, between about 1 and about 40 cycles per minute. In some embodiments, rocking can be performed periodically. Rocking can be performed continuously for a period of time (e.g., one cycle begins immediately at the conclusion of the one before). In some embodiments, rocking can be performed episodically (e.g., the slide 104 can rest for some period of time in between cycles of rocking). In some embodiments, the slide 104 can alternate between being reciprocated back and forth for contacting the conduit 108, and being rocked (or otherwise manipulated) for mixing the fluid in the capillary gap. For example, in one embodiment, the slide 104 can be reciprocated at a rate of between about 1 and 40 cycles per minute for an appropriate amount of time (such as enough time to fill the capillary gap) and then rocked for an appropriate number of cycles to mix the fluid in the capillary gap, such as between 1 and 4 cycles or more. In some embodiments, the reservoir 106 can be reciprocated to contact the conduit 108 or remain in contact with the conduit 108 long enough t o fill any voids in the capillary gap. The reservoir 106 can then be moved away from the conduit 108, and the slide 104 can then be manipulated as described to mix the fluid in the capillary gap. In some embodiments, the reservoir 106 can be eventually substantially completely emptied after a volume of fluid, such as a solvent, reagent, and/or rinse fluid, equal to the volume of the reservoir 106 evaporates from the capillary gap. In some embodiments, the reservoir 106 can be emptied by other means, such as by coupling a pressure reducer, such as a vacuum pump, to empty excess fluid within the reservoir 106. Once the reservoir 106 has been partially or completely emptied, it can be refilled with the same or a different fluid. For example, in methods requiring subsequent processing of a sample with different treatment liquids, a first fluid, such as a reagent fluid, a solvent fluid, and/or a rinse fluid, can be dispensed into the reservoir 106, and a second fluid can be dispensed into the reservoir 106 once the first fluid has been at least partially removed or emptied. The device 100 can thus limit the need for reapplication of expensive reagent fluids by providing for solvent replenishment and admixing of the solvent and reagent in the capillary gap. The reservoir 106 can be periodically refilled, such as by episodic pipette dispensing. The reservoir 106 can be either partially or completely filled with a fluid each time the fluid is dispensed. In some embodiments, the reservoir 106 can be completely filled some of the time, and only partially refilled other times. In some embodiments, the reservoir 106 can include a sensor or other monitoring device indicating the volume of fluid in the reservoir 106, or monitoring when the reservoir 106 requires refilling. Similarly, some embodiments include a sensor that can detect fluid levels in the capillary gap.

The device 100 has been described as creating a capillary gap under the slide 104. Of course, in some embodiments, the capillary gap can be created above the slide 104 (e.g., in some embodiments, the device is flipped such that the opposed surface is provided over a top surface of the slide 104, where the sample is provided on the top surface of the slide 104).

FIGS. 2-3 illustrate a top plan view of a device 200 for mitigating evaporation while treating a sample on a substrate such as a microscope slide 204. FIG. 4 shows a perspective view of the embodiment shown in FIGS. 2-3, with like reference numerals indicating like features. FIG. 9 is a cross-sectional view of the device of FIG. 2, taken along line 9-9 in FIG. 2.

The device 200 can include a slide support having a surface 202. Device 200 can also include a reservoir 206 and a conduit 208. Fluids can be introduced into device 200 through one or more inlets, ports, reservoirs, divots, or receptacles, such as the solvent dispense area 216 and the reagent dispense area 218. The slide support can be designed to accommodate a slide 204 having a sample on a surface thereof. For example, a surface 224 of slide 204 can be positioned such that it is facing (e.g., opposed to) and substantially parallel to the surface 202 of the device 200. In the embodiment of FIG. 2, the slide 204 is placed in the device 200 face down, such that a sample included on slide surface 224 is located between the slide surface 224 and the device surface 202.

Slide 204 can be maintained at a fixed height or distance above the opposed device surface 202, such as by parallel rails 226, 228. FIGS. 2-4 and FIG. 9 illustrate two parallel rails 226, 228, but other configurations for maintaining a fixed distance between slide surface 224 and the opposing surface 202 additionally or alternatively can be provided. For example, in some embodiments, only a single rail 226 is included. In some embodiments, rails 226, 228 can be arranged in other configurations, such as perpendicular to one another, or arranged at some other angle to one another. In some embodiments, more than two rails 226 can be included. In some embodiments, a single circular, elliptical, or curved rail 226 can define the fixed distance between slide surface 224 and the opposing surface 202. Any suitable combination and configuration of rails or other features (e.g., sidewalls, spacers, etc.) can be used to establish and maintain a fixed distance between slide surface 224 and the opposing surface 202. As best seen in FIG. 9, the fixed distance between slide surface 224 and the opposing surface 202 defines a capillary gap between the surfaces, in which one or more liquids 222 can be applied to the sample on the slide surface 224. In some embodiments, the reagent dispense area 218 is in fluid communication with the capillary gap between the slide surface 224 and the opposing surface 202. The reagent dispense area 218 can simply be a small reservoir or divot configured to allow any reagent dispensed into the reagent dispense area 218 to be wicked into the capillary gap via capillary action.

In some disclosed embodiments, the reservoir 206 is in fluid communication with the conduit 208 at least part of the time (e.g., the reservoir 206 can be designed to be in constant fluid communication with the conduit 208, or the reservoir 206 can be movable such that it is in fluid communication with the conduit 208 in some positions, but separated from the conduit 208 in other positions). The reservoir 206 can include a second fluid 236, such as a solvent, reagent fluid, and/or rinse fluid. The second fluid 236 can flow into the conduit 208 by virtue of capillary action when the reservoir 206 is positioned to contact the conduit 208. If the reservoir 206 is not completely filled with the second fluid 236, a void 238 can form in reservoir 206. Device 200 can be designed to ensure that the second fluid 236 remains in fluid communication with the conduit 208 despite any void 238. For example, reservoir 206 can be tilted, such as by being oriented at an angle with respect to the opposed surface 202. Alternatively, or in addition, reservoir 206 can be positioned relative to conduit 208 and designed to ensure fluid flow therebetween, such as by gravitational flow. In these embodiments, the reservoir 206 can be configured to keep any void 238 from forming near the conduit 208.

As shown in FIGS. 2-4, the conduit 208 can be arranged substantially perpendicular to the reservoir 206. In some embodiments, the conduit 208 can be positioned at an angle to the reservoir 206, such as at an angle of between zero and ninety degrees, or at an angle of between ninety and 180 degrees. The conduit 208 can be sized to be significantly smaller in length, width, and/or height as compared to the reservoir 206. In some embodiments, the height of the conduit 208 can be substantially the same as the height of at least a portion of the reservoir 206. The solvent dispense area 216 can be in fluid communication with the reservoir 206. In some embodiments the reservoir 206 can include a cover, or can be otherwise inaccessible. In such embodiments, the solvent dispense area 216 can allow for a mechanism and/or a user to add a rinse, fluid reagent, solvent, or other fluid to the reservoir 206. The slide support of device 200 can allow for movement of the slide 204 and/or movement of the reservoir 206. For example, as shown in FIG. 2, the slide 204 can be positioned in a first position such that the slide 204 is displaced, (e.g., displaced laterally), from the conduit 208. During processing of the sample, some of the fluid 222 in the capillary gap can be lost to evaporation, thus forming a void 220. To refill the void 220 and replenish the fluid 222 in the capillary gap, the slide 204 can be moved to a second position, as shown in FIG. 3. As seen in FIG. 3, the slide 204 has been moved in the direction of arrow 230 to contact the conduit 208. Specifically, the fluid 222 in the capillary gap is made to contact the second fluid 236 in the conduit 208. The second fluid 236 can then flow in the direction of arrow 232 and into the capillary gap, to combine with the fluid 222 and refill any voids 220.

The slide 204 can be moved to the second position shown in FIG. 3 in any suitable fashion. For example, the slide 204 can be translated along the rails 226, 228 towards the conduit 208, such as by an automated mechanical actuator, and/or manually by a user. After a set period of time, and/or once any voids 220 have been refilled, the slide 204 can be moved again, such as moved back to the first position as shown in FIG. 2, thus breaking the contact between the fluid 222 in the capillary gap and the second fluid 236 in the conduit 208.

In some embodiments, the slide 204 can be positioned to contact the conduit 208, and the reservoir 206 can be positioned in a first position such that the reservoir 206 is displaced, (e.g., displaced laterally), from the conduit 208. To refill the void 220 and replenish the fluid 222 in the capillary gap, the reservoir 206 can be moved to a second position in which it is brought into contact with the conduit 208. Specifically, the second fluid 236 in the reservoir 206 is made to contact the conduit 208. The second fluid 236 can then flow into the conduit 208 and into the capillary gap, to combine with the fluid 222 and refill any voids 220.

As with movement of the slide 204, reservoir 206 can be moved in any suitable fashion. For example, the reservoir 206 can be translated along rails towards the conduit 208, such as by an automated mechanical actuator, and/or manually by a user. After a set period of time, and/or once any voids 220 have been refilled, the reservoir 206 can be moved again, thus breaking the contact between the conduit 208 and the second fluid 236 in the reservoir 206.

In some embodiments, the device 200 can also include a vacuum hole 234. A pressure reducer, such as a vacuum pump, can be fluidly connected to the fluid 222 in the capillary gap through the vacuum hole 234. In some embodiments where it is desirable to apply more than one fluid 222 to the sample on slide surface 224, the vacuum hole 234 can be used to remove the fluid 222 from the capillary gap so that a new and/or different fluid can be added for application to the sample.

After a second fluid 236 has been added to the fluid 222 in the capillary gap, it can be desirable to mix the fluids in the capillary gap so that the newly added fluid is more evenly distributed. Mixing can be accomplished in any suitable manner. In some embodiments, the slide 204 can be reciprocated and/or rocked in order to mix the fluid 222 in the capillary gap. FIG. 5 illustrates a perspective view of one embodiment of a rocking mechanism 500 for mixing the fluid within the capillary gap via rocking of the slide 204. Device 500 can include a surface 502 substantially parallel to and opposing a slide surface when a slide is placed into the device 500. A slide can be inserted such that it rests on rails 526, 528, which define a fixed distance between the slide surface and the surface 502, thus creating a recess or capillary gap between the slide and the surface 502. A handle 540 can further support the slide, and a side wall 542 can assist in keeping the slide in place. The device 500 can include a dispensing area 518, such as for dispensing a fluid (e.g., a reagent) into the capillary gap.

The device 500 can, in some embodiments, include a port 544, such as a vacuum port and/or an aspiration port. Such port 544 can be used to aspirate the fluid in the capillary gap and/or pull fluid out of the capillary gap. A motor 546 can be arranged to manipulate the slide in any number of motions. For example, the motor 546 can be coupled to a cam and at least two pins 558, such that rotation of the motor 546 actuates the pins 558 up and down along a vertical axis. The pins 558 can be positioned under the slide and actuated such that one area of the slide is lifted and then another. Thus, a rocking motion can be created to encourage mixing of the fluid in the capillary gap. In the embodiment shown in FIG. 5, as positioned, pins 558 can be actuated up and down to raise and lower the left and right sides of the slide. In some embodiments, pins 558 can be positioned under different areas of the slide, such as under the top and bottom sides of the slide. In some embodiments, the motor 546 can translate the slide in the horizontal x-y plane in order to promote mixing, and/or to contact the slide to a conduit, as discussed above.

FIG. 6 is a cross-sectional view illustrating another embodiment of a device 600 for mitigating evaporation. FIG. 7 is a perspective view of the device shown in FIG. 6. As seen in FIGS. 6-7, a slide 604 can include a sample on a surface 624 thereof. A gap 656 of capillary dimensions can be maintained between the slide surface 624 and a second, opposing curved surface 648. In the embodiment illustrated in FIGS. 6-7, the opposing surface 648 is a curved plate 648. The curved surface 648 can have a radius of curvature of, for example, between about 5 inches and about 20 inches in some embodiments. A treatment fluid 622 can be provided in the gap 656.

In some embodiments, rails and/or dimples in the curved surface 648 can define the capillary gap 656 between the curved surface 648 and the slide surface 624, such as a gap having a height between about 0.003 inches and about 0.007 inches. In some embodiments, the height of capillary gap 656 can be just that of the treatment fluid 622 (e.g., the treatment fluid 622 can serve as a liquid bearing between the slide surface 624 and the curved surface 648).

The curved surface 648 can include a reservoir 606 in fluid communication with a conduit 608. The reservoir 606 can include a replenishing liquid 636, which liquid 636 can also fill the conduit 608 due to capillary action. The curved surface 648 can also include one or more additional features, such as one or more mixing troughs 650.

In use, the slide 604 can be rolled over the curved surface 648, or rocked back and forth in the directions indicated by arrows 652 and 654. Such rocking can be performed manually or in an automated manner, such as with mechanical actuation. Rocking the slide 604 in the direction indicated by arrow 654 can bring the fluid 622 to contact the fluid 636 within the conduit 608. If any voids have formed in the fluid 622 due to, for example, actuation of a pressure reducer and/or evaporation, capillary action can draw the second fluid 636 from the conduit 608 into the capillary gap 656. Rocking the slide 604 in the opposite direction along the arrow 652 can result in at least a portion of the fluid 622 entering the mixing trough 650. The fluid 622 can be forced into the mixing trough 650 by the pressure created by rocking the slide 604, and drawn out of the mixing trough 650 by capillary action when the slide 604 is rocked back in the other direction. When the fluid 622 enters and leaves the mixing trough 650, the fluid 622 can be mixed, to help evenly distribute any newly added fluid, such as fluid 636 from the conduit 608. Thus, in some embodiments, one motion (e.g., rocking the slide 604) can both provide mixing of the fluid 622 in the capillary gap and establish contact between the fluid 622 in capillary gap 656 and the fluid 636 in conduit 608. As shown in FIGS. 6-7, the mixing trough 650 can be located at a position in the curved surface 640 that is remote from the reservoir 606 and conduit 608. The device 600 can include more than one mixing troughs 650 at various locations along the curved surface 648. The mixing trough 650 can take any suitable shape or geometry, and does not necessarily have the geometry shown in FIGS. 6-7. For example, suitable mixing troughs 650 can have a substantially square, rectangular, or pyramidal cross section. Other embodiments of mixing troughs 650 can have a substantially hemispherical cross sectional area. If more than one mixing trough 650 is provided, the troughs can each have the same shaped cross sectional area, or one or more troughs may have a differently shaped cross-sectional area from one or more other troughs. In embodiments having multiple mixing troughs 650, the mixing troughs can be spaced substantially equidistant from one another along the curved surface 648. In alternative embodiments, the spacing of plural mixing troughs 650 need not be evenly distributed.

In some embodiments of devices for mitigating evaporation in a slide staining apparatus, the surface opposing the slide surface (whether curved or parallel) can be heated and/or thermally conductive. In some embodiments, devices can provide at least some thermal flow isolation of the reservoir with respect to the opposing surface. In some embodiments, the reservoir and/or conduit of the device can comprise a material having low thermal conductivity, such as, for example, a ceramic, a plastic, or combinations thereof. Devices and microfluidic features can be manufactured according to any suitable process or technique. For example, a simple glass cutter can be used to create a reservoir and/or a conduit in a flat or curved surface. Alternatively or additionally, features of an evaporation mitigation device can be, for example, machined or molded.

Embodiments of the disclosed devices can be integrated with automated systems, such as an automated slide staining apparatus. One such automated apparatus is described in U.S. Patent No. 7,303,725, which is hereby incorporated herein by reference. Other instruments that have been designed for this purpose include the Ventana Medical Systems' line of dual carousel-based instruments such as the 320/ES®, NexES®, BENCHMARK®, and the BENCHMARK® XT. Patents that describe these systems include U.S. Patent Nos. 5,595,707, 5,654,199, 6,093,574, and 6,296,809, all of which are incorporated herein by reference in their entireties. Another type of automated staining device is the TechMate® line of device, described in U.S. Patent Nos. 5,355,439 and 5,737,499, both of which are incorporated herein by reference in their entireties.

One embodiment of an automated tissue staining apparatus can comprising a slide support designed to accommodate and position a slide such that a slide surface containing a sample can be positioned facing an opposed surface of the slide support such that a capillary gap is formed between the slide surface and the opposed surface. The automated apparatus can also comprise a reagent delivery system for applying a predetermined quantity of reagent to the slide surface and a fluid delivery system for applying a predetermined quantity of fluid (e.g., a solvent) to a reservoir of the slide support. Some automated devices include a mixing system having a motor configured to reciprocate to slide, rock the slide, and/or roll the slide over a curved surface. A heat zone for heating samples positioned on the slides and at least one rinse station at a rinse zone can also be included in the automated apparatus. The rinse station can comprise, for example, at least one nozzle positioned for directing a stream of rinse liquid onto the slide. FIG. 8 illustrates a block diagram of a method for mitigating evaporation of treatment liquids from a surface. According to one method, a thin sample can be provided on a first surface, such as by providing a biological tissue sample on a microscope slide surface (step 800). A second surface (e.g., a curved or parallel surface) can be maintained opposed to and spaced by a first distance from the first surface, thereby providing a capillary gap between the first and second surfaces (step 802). A first fluid or combination of fluids, such as a reagent and solvent mixture, can be provided within the capillary gap, such as by dispensing the fluid using a micropipette (step 804). A second fluid or combination of fluids, which can be the same as or different from the first fluid, can be added to a reservoir, again, for example, by dispensing using a micropipette (step 806). In some embodiments, the reservoir can be in fluid communication with a conduit, such that when the reservoir is at least partially full, the second fluid flows from the reservoir to fill the conduit via capillary action. In some embodiments, the slide can be in fluid communication with a conduit.

Methods according to the present disclosure can comprise contacting the first fluid in the capillary gap with the second fluid in the conduit, such that the second fluid flows from the conduit into the capillary gap to fill any voids formed in the capillary gap due to evaporative loss of the first fluid (step 808). In some methods, contacting the first fluid with the second fluid can be accomplished by, for example, moving the slide from a first position to a second position in the device. In some embodiments, contacting the first fluid with the second fluid can comprise rocking or rolling the slide back and forth over a curved surface. In some embodiments, contacting the first fluid with the second fluid can be accomplished by moving the reservoir from a first position to a second position in the device.

Once contact has been established, the contact between the first liquid in the capillary gap and the second liquid in the reservoir can be broken, or removed (step 810). For example, contact can be broken by manipulating the slide and/or reservoir to move or translate it away from the conduit, such that the first and second fluids are no longer in contact. In other embodiments, contact can be broken by rolling or rocking the slide over a curved surface such that the first fluid in the capillary gap is moved to a different area of the curved surface, away from the conduit. Some methods comprise cyclic alternation between establishing and breaking contact between the first and second fluids.

Some methods comprise mixing the second fluid with the first fluid (step 812). In some methods, mixing the second fluid with the first fluid comprises rocking the slide, such as rocking the slide using a rocking mechanism. In some methods, mixing the second fluid with the first fluid comprises rocking or rolling the slide back and forth over the curved surface. In some methods, mixing can include rocking the slide over a curved surface having one or more mixing troughs. In some methods, mixing the second fluid with the first fluid comprises reciprocating the slide back and forth over the opposing surface.

At any point, any of the method steps can be repeated. For example, fluid can be dispensed into the capillary gap and/or into the reservoir multiple times during the processing of a single sample, at any point. In some methods, contacting and breaking contact between the first and second fluids can occur multiple times before and/or after mixing of the fluid in the capillary gap.

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. A device, comprising: a slide support designed to accommodate a slide having a sample on a surface thereof, the slide support including an opposed surface arranged to be opposed to and spaced apart from the slide surface to form a capillary gap; and a reservoir in fluid communication with a conduit designed to periodically replenish fluid in the capillary gap, wherein the slide support allows for movement of the slide between a first position in which a first liquid within the capillary gap is displaced from the conduit, and a second position in which the first liquid within the capillary gap is brought into contact with a second liquid within the conduit.

2. The device according to claim 1, further comprising a rocking mechanism configured to mix the fluid in the capillary gap.

3. The device according to claim 2, wherein the rocking mechanism comprises one or more pins configured to raise and lower alternating sides of the slide with respect to the slide support.

4. The device according to claim 1 , wherein the reservoir is oriented at an angle with respect to the slide.

5. The device according to claim 1, further comprising a reagent dispense area in fluid communication with the capillary gap.

6. The device according to claim 1, wherein the slide support comprises at least two parallel rails positioned between the slide surface and the opposed surface, thereby defining the capillary gap.

7. The device according to claim 1, further comprising a sensor to detect fluid levels in the reservoir.

8. The device according to claim 1, wherein the slide support allows translation of the slide in a horizontal plane.

9. The device according to claim 8, wherein the slide support allows movement of the slide in a vertical plane.

10. The device according to claim 8, wherein the slide support allows movement of the slide in the horizontal plane in a second direction, wherein the second direction is at an angle to a first direction of movement between the first and second positions of the slide.

11. A device, comprising: a slide support designed to accommodate a slide having a sample on a surface thereof, the slide support having a curved surface arranged to be opposed to the slide surface, wherein the curved surface includes a reservoir, a conduit in fluid communication with the reservoir, and a mixing trough, and wherein the slide support is designed to allow movement of the slide relative to the curved surface such that at least a portion of a liquid between the slide surface and the curved surface is moved into and out of the mixing trough as the slide is moved.

12. The device according to claim 11, further comprising at least two mixing troughs.

13. The device according to claim 11 , wherein the mixing trough is located in the curved surface at a location remote from the reservoir.

14. The device according to claim 11, further comprising a sensor to detect fluid levels in the reservoir.

15. A method, comprising: providing a thin sample on a first surface of a substrate; maintaining a second surface opposed to and spaced by a first distance from the first surface, thereby providing a capillary gap between the first and second surfaces; providing a first liquid within the capillary gap; dispensing a second liquid into a reservoir in fluid communication with a conduit, such that the second liquid fills the conduit via capillary action; contacting the first liquid in the capillary gap with the second liquid in the conduit, such that the second liquid flows from the conduit into the capillary gap to fill any voids formed in the capillary gap due to evaporative loss of the first liquid; breaking the contact between the first liquid in the capillary gap and the second liquid in the conduit; and mixing the second liquid with the first liquid.

16. The method according to claim 15, wherein mixing the second liquid with the first liquid comprises placing the slide in a rocking mechanism.

17. The method according to claim 15, wherein the steps of contacting the first liquid in the capillary gap with the second liquid in the conduit and breaking the contact are performed periodically.

18. The method according to claim 15, wherein the second surface is substantially parallel to the first surface.

19. The method according to claim 15, wherein the second surface is a curved surface.

20. The method according to claim 19, wherein mixing the second liquid with the first liquid comprises reciprocating the slide back and forth over the curved surface.

21. The method according to claim 19, wherein the curved surface includes a mixing trough.

22. The method according to claim 15, wherein the fluid tension of the first liquid in the capillary gap is greater than the fluid tension of the second liquid in the reservoir.

23. The method according to claim 15, wherein fluid tension prevents the second liquid from escaping the conduit until the second liquid contacts the first liquid in the capillary gap.

24. An automated tissue staining apparatus comprising: a slide support designed to accommodate and position a slide such that a slide surface containing a sample can be positioned facing an opposed surface of the slide support such that a capillary gap is formed between the slide surface and the opposed surface; a reagent delivery system for applying a predetermined quantity of reagent to the slide surface; a fluid delivery system for applying a predetermined quantity of solvent to a reservoir of the slide support; a mixing system having a motor configured to reciprocate the slide; a heat zone for heating samples positioned on the slide; and at least one rinse station at a rinse zone, the rinse station comprising at least one nozzle positioned for directing a stream of rinse liquid onto the slide.