WO2011008415A2

WO2011008415A2 - Cover tile for treating a substrate with a fluid

Info

Publication number: WO2011008415A2
Application number: PCT/US2010/039229
Authority: WO
Inventors: Charles D. Lemme; Glen Ward; Brian H. Kram; William L. Richards
Original assignee: Ventana Medical Systems, Inc.
Priority date: 2009-07-17
Filing date: 2010-06-18
Publication date: 2011-01-20
Also published as: WO2011008415A3

Abstract

A cover tile/substrate construct that includes a cover tile having an upper surface, an opposing lower surface, a proximal edge, a distal edge, and a longitudinal direction extending from the proximal edge to the distal edge, wherein the lower surface defines a three-dimensional surface that includes a protrusion that extends in the longitudinal direction; a substantially planar substrate having an upper surface and an opposing lower surface; and wherein the cover tile is juxtaposed over the substrate so that the lower surface of the cover tile and the upper surface of the substrate together define a wedge-shaped capillary fluid flow gap and the longitudinally extended protrusion of the lower surface of the cover tile protrudes towards the upper surface of the substrate.

Description

COVER TILE FOR TREATING A SUBSTRATE WITH A FLUID

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of and priority to U.S. Provisional Patent Application No. 61/226,610 filed on July 17, 2009, which is incorporated herein in its entirety.

FIELD

Disclosed herein are equipment and methods for preparing samples for analysis. In particular, equipment and methods are provided for automated staining of biological samples on microscope slides.

BACKGROUND

A wide variety of techniques that include placing a sample on a substrate have been developed to prepare and analyze biological samples. Examples of such techniques include microscopy, micro-array analyses (such as protein and nucleic acid micro-array analyses) and mass spectrometric methods such as MALDI and SELDI. In each of these techniques, preparation of samples for analysis can include contacting the sample on the substrate with one or more liquids. Where a sample is treated with multiple liquids, both application and subsequent removal of liquids can be important for providing a sample suitable for analysis. In the context of microscope slides bearing biological samples (such as tissue sections or cells), the sample is typically treated with one or more dyes or conjugates of specific binding agents with detectable labels (such as nucleic acid probes and antibodies labeled with enzymes or fluorescent moieties) to add color and contrast to otherwise transparent or invisible cells or cell components.

SUMMARY

Disclosed herein is a cover tile/substrate construct, comprising:

a cover tile having an upper surface, an opposing lower surface, a proximal edge, a distal edge, and a longitudinal direction extending from the proximal edge to the distal edge, wherein the lower surface defines a three-dimensional surface that includes a protrusion that extends in the longitudinal direction;

a substantially planar substrate having an upper surface and an opposing lower surface; and

wherein the cover tile is juxtaposed over the substrate so that the lower surface of the cover tile and the upper surface of the substrate together define a wedge-shaped capillary fluid flow gap and the longitudinally extended protrusion of the lower surface of the cover tile protrudes towards the upper surface of the substrate.

Also disclosed herein is a cover tile for placement over a specimen on a slide, comprising:

an elongate structure having an upper surface, an opposing lower surface, a proximal edge, a distal edge, a first lateral edge, and a second lateral edge;

a fluid inlet port extending from the upper surface of the cover tile to the lower surface of the cover tile;

a fluid outlet port extending from the lower surface of the cover tile to the upper surface of the cover tile; and

wherein the lower surface of the cover tile includes a first face that extends from the fluid inlet port to the fluid outlet port, a second face that extends from the first face to the first lateral edge, and a third face that extends from the first face to the second lateral edge; and wherein the second face and the third face are each tangential to the first face. Further disclosed herein is an apparatus for automatically applying a fluid to a substrate, comprising:

at least one cover tile having an upper surface and an opposing lower surface, wherein the lower surface defines a plurality of faces, with at least one face having a different incline relative to at least one other face;

at least one substrate having an upper surface and an opposing lower surface, wherein the cover tile lower surface is located over the upper surface of the substrate and the cover tile lower surface and substrate upper surface together define a wedge-shaped cavity;

a tray configured to hold the at least one cover tile and the substrate associated with the cover tile;

a fluid delivery mechanism for introducing a fluid into the wedge-shaped cavity; and

a fluid removal mechanism for removing the fluid from the wedge-shaped cavity. In another aspect disclosed herein is an apparatus, comprising:

a cover tile loader module that includes a moveable shuttle configured to receive a plurality of cover tiles; and

a cover tile placement module coupled to the cover tile loader module, wherein the cover tile placement module includes a first location for inserting the shuttle from the cover tile loader module; a second location for inserting a tray, the tray including a plurality of substrates; and a member positioned above the first location and the second location for removing the cover tiles from the shuttle and placing the cover tiles over the substrates in the tray. An additional apparatus disclosed herein is an apparatus for automatically treating biological specimens on a substrate, comprising:

(i) a first workstation that includes a cover tile loader module coupled to a cover tile placement module and is configured to make a plurality of cover tile/substrate constructs, wherein the cover tile is juxtaposed over the substrate so that a lower surface of the cover tile and an upper surface of the substrate together define a wedge-shaped capillary flow gap; and

(ii) a second workstation that receives the plurality of the cover tile/substrate constructs, and includes a reagent delivery mechanism for introducing a reagent into the wedge-shaped capillary flow gap and a reagent removal mechanism for removing the reagent from the wedge-shaped capillary flow gap. Also disclosed herein is an automated process for applying a fluid to a specimen disposed on a substrate, comprising:

introducing a fluid into a wedge-shaped capillary flow gap defined by a lower surface of a cover tile juxtaposed over an upper surface of the substrate, wherein the lower surface of the cover tile defines a longitudinally extended first face that protrudes toward the upper surface of the substrate and at least a second face and a third face that each laterally extend from the first face and that are angled at an incline away from the upper surface of the substrate;

allowing the fluid to initially flow along the first face via capillary action; subsequently allowing the fluid to flow along the second face and the third face via capillary action; and

then removing the fluid from the wedge-shaped capillary flow gap.

A further process disclosed herein is an automated process for making a cover tile/substrate construct, comprising:

introducing a plurality of cover tiles onto a moveable shuttle;

transporting the moveable shuttle into a cover tile placement module;

removing the cover tiles from the moveable shuttle; and

placing each cover tile over an associated substrate so that a lower surface of the cover tile and an upper surface of the substrate together define a wedge-shaped cavity.

The foregoing 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 perspective view of the lower surface of one embodiment of a cover tile disclosed herein.

FIG. 2 is a perspective view of the upper surface of one embodiment of a cover tile disclosed herein. FIG. 3 is an exploded view of a spacer element of the cover tile shown in FIG. 1.

FIG. 4 is an exploded view of a spacer element of the cover tile shown in FIG. 2.

FIG. 5 is a plan view of one embodiment of a cover tile/substrate construct disclosed herein.

FIG. 6 is a lateral cross-sectional view of the embodiment of FIG. 5 at plane AA.

FIG. 7 is a lateral cross-sectional view of the embodiment of FIG. 5 at plane BB.

FIG. 8 is a longitudinal cross-sectional view of the embodiment of FIG. 5 at plane CC.

FIG. 9 is a perspective view of the embodiment of FIG. 5.

FIG. 10 is a perspective view of a fluid volume as it occupies a wedge- shaped cavity defined by a cover tile/substrate construct.

FIG. 11 is a longitudinal cross-sectional view of a cover tile/substrate construct disclosed herein.

FIGS. 12A-12G are plan views of the upper surface of a substrate that depict the progression of fluid flow in a fluid cavity defined by the cover tile and substrate upper surface as disclosed herein.

FIG. 13 is a perspective view of a substrate tray holding cover tile/substrate constructs as disclosed herein.

FIG. 14 is a perspective view of a single cover tile/substrate construct as held in a substrate tray.

FIG. 15 is a perspective view of a receptacle area on a substrate tray for holding a cover tile/substrate construct.

FIG. 16 is a perspective view of a substrate tray holding cover tile/substrate constructs as disclosed herein.

FIG. 17 a perspective view of a substrate tray holding cover tile/substrate constructs as disclosed herein. FIG. 18 is a perspective view of a spring for holding a substrate in a substrate tray.

FIG. 19 is a perspective view of one embodiment of a reagent dispenser system as disclosed herein.

FIG. 20 is an elevation view of a fluid delivery mechanism and fluid removal mechanism as disclosed herein.

FIG. 21 is a perspective view of a cover tile loader/placement instrument as disclosed herein.

FIG. 22 is another perspective view of a cover tile loader/placement instrument as disclosed herein.

FIG. 23 is a perspective view of a cover tile loader module as disclosed herein.

FIG. 24 is an elevation view showing an initial stage of loading a cover tile.

FIG. 25 is an elevation view showing a further stage of loading a cover tile. FIG. 26 is an elevation view showing a further stage of loading a cover tile.

FIG. 27 is an elevation view showing a further stage of loading a cover tile.

FIG. 28A is a perspective view of a gripper pin as disclosed herein.

FIG. 28B is a plan view of a lower surface of a tile gripper plate as disclosed herein.

FIG. 29 is a perspective view of a cover tile placement module as disclosed herein.

FIGS. 3OA and 30B are plan views of a lower surface of a tile gripper plate as disclosed herein.

FIG. 31 is a perspective view of an upper surface of a tile gripper plate as disclosed herein.

FIG. 32 is a perspective view of one corner of a cover tile placement module as disclosed herein.

FIG. 33 is a schematic representation of a process for applying a reagent to a substrate via a capillary gap flow as disclosed herein.

FIG. 34 is a schematic representation of workstations and their process interrelationship of one embodiment of an apparatus disclosed herein. In the drawings like reference numerals refer to like elements unless otherwise indicated. DETAILED DESCRIPTION

The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. The word "comprises" indicates "includes." Any process or method described herein may be performed in any order, unless context indicates otherwise. Any numerical values recited herein include all values from the lower value to the upper value in increments of one unit provided that there is a separation of at least 2 units between any lower value and any higher value. As an example, if it is stated that the amount of a component or a value of a process variable is from 1 to 90, preferably from 20 to 80, more preferably from 30 to 70, it is intended that values such as 15 to 85, 22 to 68, 43 to 51, 30 to 32 etc., are expressly enumerated in this specification. For values, which have less than one unit difference, one unit is considered to be 0.1, 0.01, 0.001, or 0.0001 as appropriate. Thus all possible combinations of numerical values between the lowest value and the highest value enumerated herein are said to be expressly stated in this application. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, percentages, and so forth, as used in the specification or claims are to be understood as being modified by the term "about." Accordingly, unless otherwise indicated, implicitly or explicitly, the numerical parameters set forth are approximations that may depend on the desired properties sought and/or limits of detection under standard test conditions/methods. When directly and explicitly distinguishing embodiments from discussed prior art, the embodiment numbers are not approximates unless the word "about" is recited.

The following explanations of terms and methods are provided to better describe the present apparatus and methods, and to guide those of ordinary skill in the art in the practice of the present disclosure. It is also to be understood that the terminology used in the disclosure is for the purpose of describing particular embodiments and examples only and is not intended to be limiting. The term "biological reaction apparatus" refers to any device in which a reagent is mixed with or applied to a biological sample, and more particularly to any automated device that performs one or more operations on a biological sample.

The term "reagent" refers to any liquid or liquid composition used in a substrate (e.g., slide) processing operation that involves contacting a liquid or liquid composition with the substrate. Reagents include solutions, emulsions, suspensions and solvents (either pure or mixtures thereof). Reagents can be aqueous or nonaqueous. Examples of reagents include solutions or suspensions of antibodies, solutions or suspensions of nucleic acid probes, and solutions or suspensions of dye or stain molecules (such as H&E staining solutions and Pap staining solutions).

Further examples of reagents include solvents and/or solutions for de-paraffϊnization of paraffin-embedded biological samples such as limonene, aqueous detergent solutions, and hydrocarbons (for example, alkanes, isoalkanes and aromatic compounds such as xylene). Additional examples of reagents include solvents (and mixtures thereof) that can be used to dehydrate or rehydrate biological samples, such as ethanol, water and mixtures thereof.

The term "slide" refers to any substrate (such as glass, quartz, plastic or silicon) of any dimensions on which a biological sample is placed for analysis, and more particularly to a "microscope slide" such as a standard 3" X 1" glass slide or a standard 75 mm X 25 mm glass slide. Examples of biological samples that can be placed on a slide include a cytological smear, a thin tissue section (such as from a biopsy), or alternatively, can be an array of biological samples, for example a tissue array, a DNA array, an RNA array, a protein array, or any combination thereof. Thus, in one embodiment, tissue sections, DNA samples, RNA samples, and/or proteins are placed on a slide at particular locations.

The term "substrate or slide processing operation" refers to any treatment or manipulation of a slide, either with or without a biological sample already placed thereon, or any treatment of a biological sample placed on a slide. Examples of slide processing operations include, but are not limited to, cleaning, heating, cooling, drying, baking, labeling, indexing, removing mercury deposits, re-hydrating, dehydrating, fixing, de-paraffϊnizing, decalcifying, bluing, digesting, preserving, pre-stain prepping, solvent exchanging, mounting, staining and coverslipping, and combinations thereof.

The term "staining" is used herein to refer to any treatment of a biological sample (such as a cellular smear or a tissue section) that detects and/or differentiates the presence, location and/or amount (such as concentration) of a particular molecule (such as a lipid, protein or nucleic acid) or particular structure (such as a normal or malignant cell, cytosol, nucleus, Golgi apparatus, or cytoskeleton) in the biological sample. For example, staining can provide contrast between a particular molecule or a particular cellular structure and surrounding portions of a biological sample, and the intensity of the staining can provide a measure of the amount of a particular molecule in the sample. Staining can be used to aid in the viewing of molecules, cellular structures and organisms not only with bright-field microscopes, but also with other viewing tools such as phase contrast microscopes, electron microscopes and fluorescence microscopes. Some staining methods can be used to visualize an outline of a cell. Other staining methods rely on certain cell components (such as molecules or structures) being stained without staining the rest of a cell. Examples of types of staining methods include histochemical methods, immunohistochemical methods and other methods based on reactions between molecules (including non-covalent binding interactions), for example, hybridization reactions between nucleic acid molecules. Particular staining methods include, but are not limited to, primary staining methods such as hematoxylin & eosin (H&E) staining and Pap staining, enzyme-linked immunohistochemical methods and in situ RNA and DNA hybridization methods such as fluorescence in situ hybridization (FISH). Additional particular examples of staining methods can be found, for example, in Horobin and Kiernan, "Conn's biological stains: a handbook of dyes, stains and fluorochromes for use in biology and medicine," 10^th ed., Oxford: BIOS, ISBN 1859960995, 2002, and in Beesley, "Immunocytochemistry and in situ hybridization in the biomedical sciences," Boston: Birkhauser, ISBN 3764340657, 2002.

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. The microscopic slide may be made of flat glass or similar material and may be 25mm wide and 75mm long and about one mm thick. It may be symmetrical about its longitudinal axis and has a label end which is also referred to as the top end or proximal end, even though the slide typically is oriented horizontally. The end opposite the label is referred to as the bottom end or distal end. There is at least one active area on the microscopic slide on which a specimen (e.g., biopsy tissue) can be placed.

The term "workstation" refers to a position or location in a disclosed system where at least one substrate (e.g., slide) processing operation is performed, and more particularly to a modular unit inside of which one or more substrate processing operations are performed on a plurality of substrates held in a substrate tray (for example, a plurality of substrates held in a substantially horizontal position in a substrate tray). A workstation can receive a substrate tray in substantially a single position so that moveable components of the workstation can locate individual substrates within the substrate tray and precisely perform a substrate processing operation on one or more substrates in the tray (such as deliver a reagent to a particular slide or portion thereof). Examples of substrate processing operations that can be performed by a workstation include heating, drying, de-paraffmizing, pre- stain prepping, rinsing, solvent exchanging, staining and coverslipping, and combinations thereof. In some embodiments, a workstation dispenses two or more reagents to a substrate without the substrates being moved from one workstation to another during a substrate-processing operation or operations such as de- paraffmizing, staining and/or solvent exchanging. Thus, in one embodiment, a workstation includes a reagent delivery means such as a nozzle or a manifold of nozzles through which reagents are delivered to substrates held in a substrate tray, which delivery means can be moveable or fixed in position within the workstation. Thus, in contrast to some prior art "workstations" which are merely containers holding a reagent in which substrates are immersed, a workstation according to the disclosure can be an active, mechanical device that delivers reagents (such as two or more reagents) to groups of substrates held together in a substrate tray. Thus, in one aspect a work station is not a reagent bath in which substrates are immersed. In other embodiments, a workstation can include a heating element and can further include a heat directing element. A heat directing element can help to spread heat more evenly between slides held in a substrate tray. A workstation also can include one or more radiant heaters. A workstation also can include a tray tilter (such as a tilt pan) to lift one end of a substrate tray to assist with liquid removal from the tray. Alternatively a workstation can include a mechanism to tilt one or more individual substrates in a substrate tray away from a horizontal position. Workstations can further include various components that move or control other workstation components, such as stepper motors, screw drives and microprocessors. Other components that can be included in a workstation include hoses, belts, tracks, fluidics connections, metering pumps, metering valves, electrical connections, sensors and the like. In another embodiment, a workstation is a modular unit that can be interchanged between two or more positions within a disclosed system and electrically and fluidically connected to the system via a common electronics backplane and a common fluidics manifold. In yet another embodiment, a workstation can include a light source, such as a UV light source for curing an adhesive for holding a coverslip in place on a slide. Overview

In one embodiment the combination of the cover tile and the substrate upper surface defines a wedge-shaped fluid cavity where the fluid flow into and out of the cavity is driven by differential capillary forces. The distance or clearance between the substrate upper surface and the cover tile lower surface varies along the longitudinal direction and/or the lateral direction so that the fluid is initially drawn by capillary action from a proximal end of the substrate to a distal end of the substrate along the longitudinal central region of the substrate, then laterally outward and back toward the proximal end, moving any bubbles ahead of the liquid front and toward the outer edge(s) of the substrate. The wedge shape is created because the distance between the substrate upper surface and the cover tile lower surface is greater at the proximal end of the cover tile/substrate construct compared to the distance at the distal end of the cover tile/substrate construct. In other words, the cover tile may be at an incline in the longitudinal direction relative to the plane defined by the substrate upper surface.

The wedge-shaped channel for capillary flow gap has a main slope in a first direction (i.e., the longitudinal direction). Fluid is introduced through an opening where the gap is large and fluid is removed where the gap is smaller. Disclosed herein is the addition of at least one secondary slope to cause fluid to move in more than one direction. The main slope along the centerline from fluid entry to fluid exit (along the longitudinal axis of the substrate) is retained but at least one secondary slope substantially perpendicular to the main slope may be included. The secondary slope can form a shallow V-shape or U-shape so that fluid is pulled first along the centerline to the bottom of the cover tile/substrate construct, then laterally spreads out and fills toward the top of the cover tile/substrate construct, pushing any bubbles that may have formed to the outer, lateral edges of the substrate. In one

embodiment, the lower surface of the cover tile defines a three-dimensional surface that includes a longitudinally aligned protrusion. The gap between the protrusion and the upper surface of the substrate defines a very thin channel for the initial capillary flow of a fluid from a fluid inlet port to a fluid outlet port, which ports are formed in the cover tile.

A further optional slope may be added at the top, making the gap larger from the lower edge of the fluid inlet to the topmost edge of the cover tile. The further optional slope has two uses. One is to provide an additional storage volume for fluid. Fluid dispense systems always have some tolerance in the amount of volume that they deliver. The further optional slope provides some additional for the plus side of the volume tolerance. A second purpose of the further optional slope above the fluid inlet is to break, during aspiration, any meniscus that might have formed around the fluid inlet.

In one aspect, the cover tile/substrate construct takes advantage of a "double wedge" geometry resulting from the combination of the main wedge angle between the cover tile and the substrate and the sloped faces defined by the lower surface of the cover tile. The "double wedge" geometry greatly facilitates the capillary flow and bubble removal. The wedge-shaped fluid volume can provide "bias" which is a force on the fluid where surface tension pulls the fluid toward the narrower gap at the distal end of the cover tile/substrate construct. The wider gap at the proximal end may be used to quickly fill the fluid channel while the narrower gap may be used to pull the fluid to the fluid outlet port.

In addition, the configuration and placement of the cover tile enables efficient fluid introduction and removal as well as sufficient contact with the region(s) of interest on the upper surface of the substrate. For example, the cover tile/substrate construct may prevent fluid from entering areas where it could become trapped or difficult to remove. In one embodiment nothing is wetted by the fluid in the gap except the two surfaces (cover tile lower surface and substrate upper surface) that form the capillary gap. The configuration of the capillary gap may be such that bubbles are not formed, or if they are formed, they move to the perimeter of the capillary gap where they will do no harm. Furthermore, the cover tile/substrate construct may provide a variable volume under the cover tile to accommodate tolerances in the volume application process.

Cover Tile

An embodiment of the cover tile will be described in connection with FIGS.

1- 9. The cover tile 1 includes a longitudinal direction 2 and a lateral direction 3, with the length of the tile in the longitudinal direction 2 typically greater than the length of the tile in the lateral direction 3 (i.e., the cover tile typically is rectangular). The cover tile 1 also includes a longitudinal axis 4. The cover tile 1 also includes a fluid inlet port 5 and a fluid outlet port 6. The distance from the fluid inlet port 5 to the fluid outlet port may vary. The distance depends on the size of the substrate and the length of the active area. For example, the inlet port 5 may be centered at the proximal end of the active area. The outlet port 6 may be over the substrate but as close as possible to the distal end. For example, the distance may be from 30 to 70 mm, more particularly 40 to 60 mm. In one specific example, the distance is 50 mm. The inlet port and the outlet port may both be centered on the longitudinal axis 4. Alternatively, the inlet port and/or the outlet port may be centered a lateral distance away from the longitudinal axis 4. The cover tile further includes a distal edge 7 that is the farthest away from the fluid inlet port 5, a proximal edge 8 that is closer to the fluid inlet port 5, and lateral edges 9. The cover tile also defines an upper surface 10 and a lower surface 11.

The fluid inlet port 5 may be a hole having a diameter from 3 to 7 mm. In one example, the fluid inlet port 5 has a diameter of 5 mm. Too large of a diameter may leave a shadow mark since the surface tension may not retain the fluid around the inlet port 5. The fluid outlet port 6 may be a hole having a diameter from 0.5 to 3 mm. In one example, the fluid outlet port 6 has a diameter of 2 mm. Typically, the fluid outlet port 6 has a smaller diameter compared to the fluid inlet port 5. The fluid inlet port and the fluid outlet port each define individual fluid passageways that extend through the entire thickness of the cover tile. In other words, the fluid inlet port 5 and the fluid outlet port 6 each define an orifice on the cover tile upper surface 10 and a corresponding fluidly connected orifice on the cover tile lower surface 11.

The lower surface 11 of the cover tile may define a plurality of separate regions, referred to herein as "faces," each of which may have a different incline, elevation and/or topography relative to the other faces. The number of faces may range from 2 to 4. In certain embodiments, the lower surface of the cover tile defines at least three, more particularly at least four, faces. The surface defined by each face may be curved or substantially planar. The periphery of each face may define a polygonal or non-polygonal shape. The border between two contiguous faces may be a distinct line such as that formed between angled substantially planar faces or the border between two contiguous faces may be a curved region that blends one face into the other face. In the embodiment shown in FIG. 1, the cover tile lower surface defines four faces. A first face 12 is in shape of a fillet that extends in the longitudinal direction. The fillet may be centered on the longitudinal axis 4 of the cover tile. The fillet 12 may extend from the fluid inlet port 5 to the fluid outlet port 6. The fillet has a curved (e.g., a substantially circular) surface. In certain embodiments, the fillet 12 is substantially elongate and forms a cylindrical outer surface. In other embodiments, the fillet 12 has a conical shape and has a wider width at the end near the fluid inlet port 5 and tapers to a more narrow width at the end near the fluid outlet port 6. For example, the width at the fluid inlet end may be 1.5 to 3 mm, more particularly 2.2 mm, and the width at the fluid outlet end may be 0.3 to 1.0 mm, more particularly 0.5 mm.

Second and third faces 13 extend in a lateral direction from the fillet 12 and are tangent to the curved surface of the fillet. The second and third faces 13 may be symmetrical with respect to each other in geometric shape and/or angles 14.

A fourth face 15 may optionally be included that is located at a region contiguous with the fluid inlet port 5. The fourth face 15 forms a fluid accumulator region as described below in more detail. The fourth face 15 also is disposed at a slope relative to the surface plane of the fillet 12 and the surface planes of the lateral faces 13.

The cover tile can be made from any material that is suitable for the operating conditions of the reagent action processes and that does not hinder the desired fluid dynamics. Illustrative materials include polyethylene (e.g., HDPE) and polyetherimide. In one embodiment, the cover tile is a singular molded object. For example, the cover tile may be made from an injection molded inert plastic (which may be filled with an inert filler such as glass beads) that has low shrinkage. Low shrinkage is conducive to molding parts with minimal warping. Inertness may be useful to minimize degradation by the reagents used in the staining process, such as organic solvents like ethanol or limonene. Cover Tile/Substrate Construct

The cover tile is disposed over a substrate 40 to form a cover tile/substrate construct 45. According to certain embodiments, the substrate 40 may be a substantially flat substrate (e.g., a slide) as described above. The substrate 40 includes an upper surface 41 and an opposing lower surface 42. The substrate may also include a label 43 (e.g., a bar code) for identification purposes. The cover tile may cover substantially all, or only portion of, the substrate upper surface 41. For example, the cover tile may be centered over the substrate and optionally extend over the lateral edges of the substrate. The cover tile may also extend beyond the distal and/or proximal end of the substrate. In one embodiment, the cover tile 1 is aligned with the underlying substrate 40 so that the fluid inlet port 5 is at the top of the active area on the substrate.

A wedge-shaped cavity 44 is defined by the cover tile lower surface 11 and the substrate upper surface 41. The wider dimension 48 of the wedge-shaped cavity is located at or near a proximal end 46 of the cover tile/substrate construct 45. The narrower dimension 49 of the wedge-shaped cavity is located at or near the distal end 47 of the cover tile/substrate construct 45. Thus, the cover tile 1 is positioned at an incline in the longitudinal direction 2 relative to the plane defined by the substrate upper surface 41. In an embodiment with a substantially flat substrate, the shape of the cavity 44 is solely determined by the cover tile lower surface 11 topography and the angle of incline between the cover tile and the substrate. In a preferred embodiment the substrate 40 is horizontal; however, the substrate 40 is not required to be horizontal.

The distance at gap 50 between the lower surface of the fillet 12 and the substrate upper surface 41 at or near the proximal end 46 may be 300 to 600 μm, more particularly 400 to 500 μm. The distance at gap 51 between the lower surface of the fillet 12 and the substrate upper surface 41 at or near the distal end 47 may be 30 to 200 μm, more particularly 75 to 175 μm. The construct may have a gap ratio of at least 2:1 to 4:1, more particularly 3 : 1 , along the longitudinal axis 4 of the cover tile 1. In other words, gap 50 is 2-4, more particularly, 3 times greater than gap 51. As described above, the cover tile lower surface 11 includes a central fillet 12 and two lateral faces 13,14 that slope upwardly from the central fillet 12. The gap between the central fillet 12 and the substrate upper surface 41 is smaller compared to the gap between the cover tile lower surface 11 and the substrate upper surface 41 at the lateral edges 8 of the cover tile 1. For example, the lateral edge gap height 52 at or near the proximal end 46 of the cover tile/substrate construct may be 75 to 200 μm, more particularly 100 to 175 μm greater than the height of gap 50. The lateral edge gap height 53 at or near the distal end 47 of the cover tile/substrate construct may be 50 to 125 μm, more particularly 75 to 100 μm greater than the height of gap 51. In one example, gap 50 is 450μm, gap 51 is 150μm, and the change in height between gap 50 and lateral edge gap 52 (referred to as the lateral delta in height) is 150μm and lateral delta height between gap 51 and lateral edge gap 53 is lOOμm. The width over which the lateral delta height rises is 1 lmm (i.e., the lateral width of the substrate over which each of the second and third faces are spaced, respectively) meaning that that second and third faces form an angle relative to the upper surface of the substrate that is arctan (0.100/11) = 0.52° (at the distal end) and arctan (0.150/11) = 0.78° (at the proximal end). In general, the angle may range from 0.25° to 0.65° at the distal end and 0.55° to 0.90° at the proximal end.

In one embodiment, the peripheral gaps at the edges of the cover

tile/substrate construct are exposed to air. The peripheral edges of the cover tile may extend beyond the peripheral edges of the substrate (e.g. the distal end of the cover tile may extend beyond the distal end of the substrate by 3mm and the cover tile is 3 lmm wide for a 25mm wide substrate). The liquid is retained between the cover tile and the substrate due to the formation of a concave meniscus at the liquid- to-air boundary as a result of surface tension. The negative radius of the concave meniscus produces a negative pressure in the liquid that retains the liquid against the gravity forces tending to pull the liquid out from between the cover tile and the substrate.

The cover tile lower surface and the substrate upper surface also define an accumulator region 54. The accumulator region 54 can serve as a reservoir that can hold an overfill of fluid. The longitudinal distance 24 of the fluid volume 55 from the lower edge of the fluid inlet part 5 to the distal end 47 of the substrate 40 may be 40 to 60 mm. The longitudinal distance 25 of the fluid volume 55 from the upper edge of the fluid inlet part 5 to the distal end 47 of the substrate 40 may be 45 to 65 mm.

The cover tile may be spaced off of the substrate by posts 18 that contact the upper surface 41 of the substrate so that the size of the gap is independent of the thickness of the substrate. Recesses 19 may be included in the top side of each post 18. These recesses 19 are designed to mate with the posts 18 of another cover tile, so that the cover tiles may be stacked on top of each other, forming a vertical column. The posts 18 may be permanently affixed to the cover tile 1 or they may be permanently affixed to a tray 75 (tray 75 is described below). In one embodiment the posts 18 are affixed to the cover slide 1 via cantilevered beams 21 that extend from the cover tile. The beams 21 typically are located near the proximal 8 and distal 7 ends of the cover tile. Shorter posts 20 extend off of beams 21 near the distal end 47 of the cover tile/substrate construct. Longer posts 22 extend off of the bottom of the beams 21 near the proximal end 46 of the cover tile/substrate construct. The lengths of the shorter posts 20 and the longer posts 22 set the gap that their respective ends, with the gap near the proximal end being larger than the gap at the distal end. The posts 18 may be spherical in shape where they contact the substrate 40 with a radius of about one mm. Alternatively, the spacer posts may be inverted and attached to a substrate tray (described below in more detail). In this embodiment, beams can extend laterally from the cover tiles to set on top of the posts.

The dimensions of the gaps 48, 49, 50, 51, 52 and 53 may be adjusted depending upon the reagent and/or the length of the active area. The gap dimensions may be adjusted by adjusting the height of the spacer posts 18 and/or by employing cover tiles having different lower surface profiles.

FIG. 10 depicts an exaggerated representation of the shape of a fluid volume 55 fully occupying the wedge-shaped cavity 44. The surface of the fluid volume 55 is the reverse of the shape of the lower surface 11 of the cover tile 1. The total fluid volume of the cavity 44 between the cover tile 1 and the substrate 40 may range from 200 to 600 μl, more particularly 425 to 525 μl. When 446 to 524 μl is filled into the cavity 44, the nominal distance of the fluid may be set to be at the center of the inlet port 5. If there is under fill, the fluid will still reach the lower edge of the inlet port 5 and cover substantially all of the active area. If there is over fill, fluid flows up into the accumulator region 54.

In one embodiment, the cover tile may include beveled corners 17. For example, the cover tile may have at each of its four corners a 45° chamfer from which extend the cantilevered beams 21. The lower surfaces 23 of the beams 21 are nominally one mm vertically above the wetted lower surface 11 of the cover tile 1. The transition from the wetted surface to the lower surface 23 of the beams 21 is sharp, forming a one mm high jump across which fluid cannot pass. This configuration keeps the posts 18 dry.

The lower surface 11 of the cover tile may be treated to enhance the capillary flow. For example, the wettability of the lower surface 11 may be increased by coating the surface with polytetrafluoroethylene or other highly hydrophobic materials.

Cover Tile/Substrate Workstation The cover tile/substrate construct may be retained in a substrate tray 75 as shown in FIGS 13-18. The substrate tray 75 may be a component of a slide processing system as described, for example, in U.S. Patent No. 7,468,161, which is incorporated herein by reference. The substrate tray 75 typically is rectangular and includes side walls 81,82, a bottom and an upper surface 84. The substrate tray 75 includes at least one, and preferably a plurality of, receptacles 85 for holding the substrates. Each individual receptacle 85 can hold a single substrate. The substrate tray can hold plurality of substrates 40 in a substantially horizontal position in the same plane. Holding all the slides in separation and in essentially the same substantially horizontal plane facilitates baking and drying can prevent cross- contamination of slides during de-paraffmizing, staining, washing and solvent exchanging, and other steps that involve dispensing reagents to slide surfaces. An optional splash guard can be added to further inhibit transfer of reagent from one slide to another. The substrate tray 75 can be used for automated handling of a plurality of substrates through of the steps of drying/baking, de-paraffinizing, staining and coverslipping using workstations configured to treat the slides as they are held in the tray's particular configuration. In the embodiment of FIG. 13, substrate tray 75 is configured to accommodate 18 specimen slides arranged in a generally horizontal grid of two rows of slides, each of which rows contain eight slides. However, the substrate tray could be configured to hold any number of substrates. The tray 75 may also include a protrusion 83 extending from a side wall 81 or 82 that can be engaged by a transporter as the substrate tray is moved within the disclosed system. The protrusion 83 may be formed from a soft iron bar embedded in the plastic of the tray 75.

Each substrate receptacle 85 is defined by guide(s) and fastening element(s) for positioning and retaining the substrate 40, and by at least one opening 86 formed in the substrate tray 75. For example, the substrate tray 75 includes a front substrate guide 76 and a rear substrate guide 77 for positioning the substrate. The guides 76,77 may be walls or partitions extending from the upper surface 84 of the substrate tray 75. The guides are positioned at dimensions that match the outer dimensions of the substrate. The substrate 40 is urged downward against the tray by a spring 78 (shown in FIG. 18 as a standalone element). The spring 78 is retained within opening 86. Clamps 79 retain the cover tile 1, locating it laterally in the horizontal plane of the substrate 40 and urging the cover tile 1 downward against the substrate 40. The cover tile 1 is aligned in the longitudinal direction by stops 80 formed on the tray 75.

Additional features of substrate tray 75 not shown in the figures could include a magnet that can be used in conjunction with one or more Hall-effect sensors placed in one or more locations (such as in one or more workstations) in the disclosed system to detect when the substrate tray occupies those locations. Tabs and indents at the corners of the tray can be used to stack several trays on top of one another without the slides touching so trays can be stored without taking up more laboratory bench space than necessary. Substrate tray 75 can be constructed of any material including a metal (such as aluminum, magnesium or a lightweight metal alloy) or a plastic (such as ABS or a thermoplastic), and can be formed, for example, by machining, casting or molding.

The substrates 40 and cover tile 1 may be placed in the substrate tray 75 manually or automatically. The substrates may be manually loaded onto the substrate tray 75, for example, at a microtome station while the substrates are still wet. The substrate trays may then be dried and substrate presence is detected and the corresponding bar codes read. The substrate trays 75 can then be manually loaded onto a cover tile placement apparatus as described below.

One embodiment of an automatic cover tile loading/placement instrument

100 is shown in FIGS. 21-31. The instrument 100 includes an automatic loader module 175 and a cover tile placement module 104. The instrument 100 may be used to place the cover tiles 1 in the proper position and to remove the cover tiles 1 after use. The instrument 100 may include a compartment that may include walls, a top and a bottom. For simplicity purposes, only the right side wall 101 is depicted in the figures. A cover tile shuttle 102 may be used to arrange and load the cover tiles 1 in positions corresponding to a respective substrate 40. As shown in detail in FIGS. 21, a stack 103 of cover tiles 1 is disposed on the outside of the right side wall 101. A cover tile placement module 104 is positioned on the inside of the loader compartment. The right side all 101 includes an opening 111 through which the cover tile can pass. The cover tile placement module 104 includes a frame having a top panel 105 and a bottom panel 106. Disposed between the top panel 105 and the bottom panel 106 is a movable tile gripper 107. The bottom panel 106 is configured to support a substrate tray 75. The tile gripper 107 is located above the substrate tray 75. A shuttle motor 108 is coupled to the cover tile shuttle 102 to enable the desired movement of the shuttle via a shuttle motor lead screw 112. The shuttle includes a platform 114 that supports two rows of shuttle pockets 113, each pocket 113 being configured to receive a cover tile 1 as described below.

As shown in FIGS. 24-27, cover tiles 1 are lowered into shuttle positions with two sets of ball bearings 109 that can be raised and lowered by air cylinders 110. The total vertical movement of the ball bearings 109 corresponds with the cover tile thickness (typically 0.050 inch). As shown in FIG. 25, a cover tile 1 is lowered into a shuttle pocket 113 and then laterally transported on the ball bearings 109 in the direction of the cover tile placement module 104 via actuation of the shuttle motor lead screw 112. As the cover tile moves farther, one of the ball bearings 109 extends upwards to support the cover tile column (see FIG. 26). As shown in FIG. 27 the cover tile 1 continues to move and then the other bearing 109 is extended to support the cover tile column. The loader 100 is now in position to receive the next cover tile 1. The cover tile loader 100 can load tiles only in positions corresponding to substrate-occupied positions in the substrate tray 75. The cover tile loader 100 also can load tiles while waiting for insertion of the next substrate tray 75.

The cover tile shuttle 102 moves the cover tiles into position over the substrate tray 75 as described below in connection with FIGS. 28-32. A loaded shuttle 102 (i.e., the shuttle platform 114 with cover tile-occupied shuttle pockets 113) moves into the cover tile placement module 104. The shuttle platform 114 with empty shuttle pockets 113 is withdrawn after the tile gripper grips and raises the tiles so that the tile can then be placed onto the substrate tray 75. In general, the tile gripper can load or unload all cover tiles on a substrate tray simultaneously, grip and lift cover tiles from the shuttle, lower and insert cover tiles into substrate positions on the substrate tray, grip and lift/extract used cover tiles from the substrate tray, and drop used cover tiles onto a passive slope for disposal. In particular, the tile gripper 107 may be a flat plate that includes an upper surface 115 and a lower surface 116. An array of gripper pins 117 protrude from the lower surface 116 of the tile gripper 107. The gripper pins 117 include a cylindrical shaft 118 and a flange 119 attached to the shaft 119 and that is elongated in a direction perpendicular to the long axis of the cylindrical shaft 119. According to one embodiment, there are four gripper pins 117 per cover tile 1. As shown in FIG. 29, the tile gripper 107 is lowered over the top of the shuttle platform 114 that includes the cover tiles 1 (or over the substrate tray that includes used cover tile/substrate constructs 45). An air cylinder 120 positioned on the top panel 105 of the frame can be used to raise and lower the tile gripper 107. The tile gripper 107 is secured to the cover tile placement module 104 frame via an attachment to a linear bearing 121 which is coupled to a linear shaft 122. There are four linear bearing 121 /linear shaft 122 assemblies, one each at the corner of the top panel 105 and lower panel 106. The linear shaft acts as a support that spaces the top panel 105 off of the lower panel 106. After the tile gripper 107 is lowered over the cover tiles 1, the gripper pins 117 are rotated so that the flange 119 engages with, and clips to, the lower surface 111 of the cover tiles 1 (see FIGS. 30A and 30B). A spring plunger 123 is located at the center of each cover tile 1 to hold the tiles in place when they are raised, lowered, or held waiting for the substrate tray 75 to be loaded in the module 104. The gripper pins 117 are rotated using a pair of racks 124 and a pinion gear 125 for each pin. An air cylinder 126 moves the racks 124 to rotate the pinion gears 90° (see FIG. 31). Conical locating posts 127 position the shuttle and the substrate tray accurately as the gripper plate 107 is lowered. Use of Cover Tile/Substrate Construct

The cover tile/substrate construct can be used for staining or other types of liquid reagent-induced analysis of specimens. The substrates 40 are placed into the tray 75. The substrates 40 are guided & located by the guides 76,77 and urged against the tray 75 by wire springs 78. The cover tiles 1 may be are automatically installed over the substrates 40 by a robot (as described above). The cover tiles 1 are juxtaposed against the substrates 40 via the spacer posts 18 thereby forming a wedge-shaped capillary gap that is larger at the label or top end of the substrate 40 and smaller at the opposite end, and larger at the lateral edges of the substrate 40 than at the center.

Staining or other types of reagent use generally involves flowing a fluid into the capillary gap between the cover tile 1 and the substrate 40, and then allowing the fluid to incubate for a sufficient period of time to interact with the specimen. FIGS. 12A-12G depict the fluid flow progression. Fluid is introduced into the inlet port 5. In one embodiment, gravity causes fluid to enter the inlet port 5. The inlet port 5 may be located over the portion of the gap where the gap is at, or near, its maximum height so that the capillary forces pull the entering fluid down the thin flow channel (defined as the area (e.g., see gaps 50 and 51) between the substrate upper surface 41 and the central fillet 12) in the direction of the smaller gap. When fluid passes through the inlet port 5, it enters the thin flow channel gap and is immediately pulled by capillary forces toward the narrower end. FIGS. 12B-12D show the progression of the fluid front 87 in the longitudinal direction 88. Once the fluid reaches the end of the thin flow channel at distal end of the substrate, the fluid then begins to flow in a lateral direction 89 toward the lateral edges of the substrate (see FIG. 12E). The fluid also begins to flow in a counter-longitudinal direction 90 toward the proximal end of the substrate (see FIG. 12F). The lateral flow 89 and counter-longitudinal flow 90 occurs down the slopes and along the surfaces of the lateral faces 13. FIG 12G shows the fluid wetting almost the entire volume of the gap between the cover tile 1 and the substrate 40.

Any bubbles 91 formed in the fluid are driven toward the fluid front as it progresses through the capillary gap. These bubbles 91 then are pushed laterally towards the peripheral edges of the substrate so that they will not interfere with the interaction between the fluid and active area of the substrate (see FIGS. 12E- 12G).

After the gap volume is filled, the fluid is allowed to remain in the gap for the desired incubation time (e.g., sufficient time to produce a stain). The cover tile/substrate construct can also be heated if desired to facilitate the reagent action. The incubation times may vary widely depending upon the particular reagent and the specimen, but in general may be 15 seconds to 15 minutes, more particularly 30 seconds to 8 minutes. At the end of the incubation time, the fluid may be evacuated from the capillary gap by lowering an elastomeric vacuum cup (see FIG. 20) down onto the fluid outlet port 6. A partial vacuum is applied which draws the fluid below the cover tile up into a vacuum line. Fluid in the capillary gap that is above the fluid outlet port 6 then flows toward the fluid outlet port 6 because of the differential capillarity of the wedge shaped gap. After the fluid is removed from the capillary gap, a new fluid, often a rinse fluid, is added to the fluid inlet port 5 to again fill the gap volume. This process is repeated with various fluids until the desired stain is produced. A hematoxylin/eosin ("H&E") stain process was performed using a cover tile/slide construct to implement exposure to a series of reagent applications. Since any given volume of reagent is distributed over the surface of the slide with a gap height that varies depending on relative x-y position (i.e. an x-y axis coordinating to the plane of the upper surface of the slide), the reagent mass exposure for a particular point within the slide specimen varies with respect to other points. Thus, a sloping fluid wedge suggests potential for variable mass exposure with consequent variable or gradient staining. Such stain variability may be averted by avoiding processing conditions resulting in relative mass depletion in those regions with the smallest gap height. It is noted that mass depletion is moderated by dye absorption kinetics at the surface of the specimen as well as by diffusivity of the particular reagent dye of interest. Therefore, relative mass depletion may be avoided for a given minimum gap height by not allowing too much exposure time to pass for a given reagent application. It was determined that 0.006" minimum gap height worked well for 3 minute hematoxylin exposure, for example. Eosin appeared to require a shorter exposure time for the same gap height.

Substrate Processing Systems

The substrates disclosed herein can be processed by any system that is suitable for analyzing samples disposed on the substrates. Such systems are described, for example, in U.S. Patent No. 7,468,161, which is incorporated herein by reference.

In one aspect, the disclosed system can include one or more workstations where biological samples on slides can be subjected to various treatments including drying, baking, de-paraffϊnizing, pre-stain prepping, staining, coverslipping and sealing, and combinations thereof. A transporter also is included for moving a slide tray carrying a plurality of slides between the plurality of workstations.

Additionally, a fluidics module, a pneumatics module and a control module can be included to deliver reagents, deliver vacuum and/or pressurized gas, and coordinate function of system components, respectively. In a particular working embodiment, the disclosed system includes a plurality of workstations that are arranged in a vertical stack and a transporter that comprises an elevator configured to move a slide tray between the vertically arranged workstations and an X-Y shuttle table configured to move a slide tray horizontally, such as in and out of a workstation, in and out of the system itself, or in and out of a parking garage. Particular examples of workstations that can be included in the system are a baking or drying station, a de-waxing or de- paraffinizing station, one or more staining stations and a coverslipping station. In a more particular embodiment, a workstation is provided that can perform two or more of de-paraffinizing, staining and solvent exchanging. In even more particular embodiments, such a workstation has a moveable nozzle assembly configured to deliver reagents to individual slides held in a slide tray. Workstations according to the disclosure can be modular and include common electrical, pneumatic and fluidic interfaces such that workstation can be easily added or removed to any of several positions within a slide processing system.

In another aspect, a fluidics module is disclosed for automated handling of reagents that can deliver reagents in packaged concentration or in diluted

concentration to a workstation without the need to disrupt the delivery of such reagents by the workstation while replacing or replenishing reagents to the system. In more particular embodiments, the fluid-handling module includes a dual chamber fluid pump. The dual chamber fluid pump includes a pump chamber and a dispense chamber where the pump chamber is configured to alternate between vacuum and pressure. The two chambers and a set of valves allow the dispense chamber to be maintained at a constant pressure for dispensation of a reagent to slides even while additional reagent is added to the dispense chamber from the pump chamber.

Alternatively, a pump chamber supplying a dispense chamber can further function as a dilution chamber, and a concentrate pump chamber can be added to provide concentrated solutions to the dilution chamber.

The cover tile/substrate constructs and cover tile loading workstation described herein can be used as a component of any of the processes described in U.S. Patent No. 7,468,161. For example, in one embodiment, the process sequence includes drying/baking, slide detect/slide position detect, cover tile placement/cover tile position detection, staining, cover tile removal, and then cover slipping. The possible process steps are schematically represented in FIG. 34.

In one embodiment, the disclosed system includes the substrate tray 75 holding a plurality of cover tile/substrate constructs in a substantially horizontal position and a workstation that receives the substrate tray 75. In a particular embodiment, a workstation delivers a reagent to upper surfaces 41 of the substrates 40 without substantial transfer of reagent (and reagent borne contaminants such as dislodged cells) from one substrate to another. In another particular embodiment, the substrate tray holding the plurality of substrates holds two or more rows or banks of substrates, for example, two rows of 4-10 substrates each.

In a more particular embodiment, the cover tile/substrate constructs are held in a rectangular substrate tray in two rows such that their long dimensions are disposed outward from the central, long axis of the tray toward the long edges of the tray. A reagent dispenser in a workstation is positioned above one or more pairs of cover tile/substrate constructs in the opposite rows, and delivers a reagent to the fluid inlet port 5 of one or more cover tiles 1 in one or the other of the two rows, for example, to a pair of substrates that are opposite from each other in the two rows. If the reagent dispenser is positioned above fewer than the total number of substrates that are held in the tray, the reagent dispenser can move to dispense reagent to other substrates in each row of substrates, and/or the substrate tray can be moved to bring additional substrates into position for reagent dispensing. Alternatively, two or more stationary or moving reagent dispensers can be included in the workstation, or one or more manifolds of dispense nozzles can be positioned above the two rows of cover tile/substrate constructs, for example, along the central, long axis of the tray.

In another particular embodiment, a workstation includes two or more sets of nozzles that are formed or inserted into a movable block that can be moved along the central, long axis of the tray to dispense reagents to one or more substrates, for example, a pair of substrates disposed toward opposite sides of the tray. Since substrates are held in the substrate tray so that they are not touching each other, and the substrates are held parallel to one another along the direction in which a reagent is dispensed from the nozzles, reagent applied to one substrate has a minimal or substantially non-existent chance of reaching another substrate and thereby cross- contaminating the substrates.

One example of a reagent dispenser system 150 is shown in FIGS. 19-20. The reagent dispenser system of FIG. 19 includes a top portion (not shown for clarity) and a bottom portion 159 that form a compartment that receives a substrate tray 75 and is configured to perform one or more slide processing operations. A nozzle manifold 160 includes a pair of fluid delivery nozzle heads 152. The nozzle manifold 160 is attached to first rail 157. The nozzle manifold 160 can be moved along first rail 157 within the workstation by stepper motor 155 coupled to a screw drive (not shown). Reagents are supplied to nozzle manifold 160 through fluid delivery conduits (e.g., tubing) (not shown) that are directed through an energy chain 151 so that the tubing does not interfere with the movement of the nozzle manifold 160 over successive pairs of substrates in substrate tray 75. The fluid delivery conduits are fluidly coupled to a fluidics module. An example of a fluidics module is schematically represented in FIG. 33.

In one embodiment, the individual fluids are individually plumbed to the energy chain 151 (in other words, each fluid has its own dedicated delivery conduit). The fluid delivery nozzle head 152 may include a single fluid dispense element 154 that may be movable from one fluid delivery conduit to another fluid delivery conduit. Alternatively, the fluid delivery nozzle head 152 may be fluidly coupled to at least one dedicated fluid delivery needle, which fluid delivery needle is fluidly coupled to only one fluid delivery conduit. In certain embodiments, the fluid delivery needle may be the same structure as the fluid delivery conduit (e.g., plastic tubing). The fluid dispense element 154 or fluid delivery needle is located so that it can be successively aligned with the fluid inlet ports 5. In one embodiment, there is a 3mm clearance between the bottom of the fluid delivery needle and the upper surface of the cover tile. In another embodiment, the same 2mm OD tubing that delivers the reagents from the valves and through the energy chain may be used as the fluid delivery nozzle. The tubing (e.g., PTFE) has minimal affinity for the reagents so that when the valve closes the momentum of the fluid stream pulls the fluid stream, breaking it up within the tubing a couple of mm from the fluid delivery exit. The end of the tubing may be cut square and retained in a clamping device with its exit end 3mm vertically above the upper surface of the cover tile. When the valve closes, the flow quickly decelerates but the fluid stream still has momentum and the portion near the exit of nozzle continues to flow, breaking off inside the tubing about 2 mm from the end. This is a surprising effect and beneficially eliminates the possibility of hanging drops.

The various individual reagent supplies and the air supply to the energy chain 151 via a series of conduits and valves (not shown). The valves can be operated to control the fluid flow into the cover tile/substrate construct 45. Selection can be performed under computer control. In some circumstances, more than one reagent can be introduced into the same line (continuously or in pulses) to provide mixtures of reagents, for example, deionized water/alcohol mixtures, and mixing chambers (such as inline mixing chambers) can also be included. Note that at least some of the nozzles on the two sides of the nozzle assembly are separately plumbed, making it possible to apply a reagent to only one slide in a pair of slides on opposite sides of a slide tray. Thus, a reagent can be applied to two slides in an opposed pair in series or simultaneously. Or, if no slide was detected in a position in a tray, no reagent need be applied to that position while a slide in an opposed position can be treated. In other embodiments, each different type of nozzle in a nozzle assembly can be separately plumbed or all nozzles of a particular type can be plumbed together.

The system 150 also includes an aspiration head 153. The aspiration head 153 is fluidly coupled to a pump system (not shown). The aspiration head 153 is attached to second rail 158. The aspiration head 153 can be moved along second rail 158 within the workstation by stepper motor 156 coupled to a screw drive (not shown). The aspiration head is aligned so that it can be successively aligned with the fluid outlet ports 6. In the embodiment shown in FIG. 20, the aspiration head 153 is attached to a suction cup 161 that is movable in the vertical direction so that the cup 161 can be lowered over a fluid outlet port 6. The disclosed cover tile system may also have several additional noteworthy features. For example, the regent dispenser (i.e., stainer) does not require rinse nozzles, purge manifolds or tipping degrees of freedom. However, the stainer may optionally include rinse nozzles, purge manifolds and/or tipping degrees of freedom. In addition, means for leveling the cover tile/substrate construct are not required (although may optionally be included) since the capillary flow enabled by the presently disclosed apparatus still occurs with a substrate tilted up to an angle of 30° from horizontal.

Several illustrative embodiments are described below in the following numbered paragraphs :

1. An apparatus for automatically applying a fluid to a substrate,

comprising:

at least one substrate having an upper surface and an opposing lower surface, wherein the cover tile lower surface is located over the upper surface of the substrate and the cover tile lower surface and substrate upper surface together define a wedge- shaped cavity;

a tray configured to hold at the least one cover tile and the substrate associated with the cover tile;

a fluid removal mechanism for removing the fluid from the wedge-shaped cavity.

2. The apparatus of paragraph 1, wherein the cover tile further includes a fluid inlet port and the fluid delivery mechanism includes at least one fluid delivery nozzle aligned with the fluid inlet port. 3. The apparatus of paragraph 1 or 2, wherein the cover tile further includes a fluid outlet port and the fluid removal mechanism includes a suction cup aligned with the fluid outlet port. 4. An apparatus, comprising:

a cover tile placement module coupled to the cover tile loader module, wherein the cover tile placement module includes a first location for inserting the shuttle from the cover tile loader module; a second location for inserting a tray, the tray including a plurality of substrates; and a member positioned above the first location and the second location for removing the cover tiles from the shuttle and placing the cover tiles over the substrates in the tray. 5. The apparatus of paragraph 4, wherein the cover tile loader module includes at least one sleeve for holding a stack of cover tiles, and the moveable shuttle includes a shuttle platform that supports a plurality of pockets, each pocket being configured to receive a cover tile from the sleeve as the shuttle moves. 6. The apparatus of paragraph 4 or 5, wherein the cover tile placement module includes a top panel, a lower panel, and a plurality of shafts connected to, and between, the top panel and the lower panel, the top panel and lower panel being sufficiently spaced apart to receive the shuttle, the tray and the member. 7. The apparatus of paragraph 6, wherein the member is a substantially flat plate that defines an upper surface and a lower surface and that includes a plurality of pins extending from the lower surface of the flat plate, and the pins include a flange configured to releasably grip a peripheral edge of a cover tile. 8. The apparatus of paragraph 7, wherein the member is slidably engaged with the top panel and the shafts. 9. An apparatus for automatically treating biological specimens on a substrate, comprising:

(ii) a second workstation that receives the plurality of the cover tile/substrate constructs, and includes a reagent delivery mechanism for introducing a reagent into the wedge-shaped capillary flow gap and a reagent removal mechanism for removing the reagent from the wedge-shaped capillary flow gap.

10. The apparatus of paragraph 9, further comprising at least one additional workstation configured to perform at least one process selected from drying, baking, de-paraffϊnizing, pre-stain prepping, coverslipping or sealing.

11. The apparatus of paragraph 9 or 10, further comprising a transporter system for transporting the substrates from one workstation to another workstation.

12. An automated process for applying a fluid to a specimen disposed on a substrate, comprising:

allowing the fluid to initially flow along the first face via capillary action; subsequently allowing the fluid to flow along the second face and the third face via capillary action; and then removing the fluid from the wedge-shaped capillary flow gap.

13. The process of paragraph 12, wherein the fluid is a reagent and the process further comprises incubating the specimen on the substrate for a

predetermined period of time prior to removing the fluid from the wedge-shaped capillary flow gap.

14. The process of paragraph 12 or 13, wherein removing the fluid from the wedge-shaped capillary flow gap comprises aspirating the fluid from the wedge- shaped capillary flow gap.

15. The process of any one of paragraphs 12 to 14, wherein introducing the fluid into the wedged-shaped capillary flow gap comprises introducing the fluid into a fluid inlet port provided in the cover tile and removing the fluid from the wedge - shaped capillary flow gap comprises removing the fluid through a fluid outlet port provided in the cover tile.

16. An automated process for making a cover tile/substrate construct, comprising:

introducing a plurality of cover tiles onto a moveable shuttle;

transporting the moveable shuttle into a cover tile placement module;

removing the cover tiles from the moveable shuttle; and

17. The process of claim 16, wherein the cover tile placement module includes a substantially flat gripper plate that defines an upper surface and a lower surface and that includes a plurality of pins extending from the lower surface of the flat plate, and the pins include a flange configured to releasably grip a peripheral edge of a cover tile, the process further comprising actuating the gripper plate and the pins to grip the peripheral edge of the cover tile.

In view of the many possible embodiments to which the principles of the disclosed apparatus and methods may be applied, it should be recognized that the illustrated embodiments are only preferred examples and should not be taken as limiting the scope of the invention.

Claims

What is claimed is:

1. A cover tile/substrate construct, comprising:

2. The construct of claim 1, wherein the substrate is a slide that includes at least one specimen.

3. The construct of either claim 1 or 2, wherein the cover tile further includes a fluid inlet port extending from the upper surface of the cover tile to the lower surface of the cover tile, and a fluid outlet port extending from the lower surface of the cover tile to the upper surface of the cover tile, and wherein the longitudinally extended protrusion extends from the fluid inlet port to the fluid outlet port.

4. The construct of any one of claims 1 to 3, wherein a gap distance between the lower surface of the cover tile and the upper surface of the substrate at the proximal edge of the cover tile is greater than a gap distance between the lower surface of the cover tile and the upper surface of the substrate at the distal edge of cover tile.

5. The construct of claim 3 or 4, wherein the fluid inlet port is near the proximal edge and the fluid outlet port is near the distal edge.

6. The construct of claim 4 or 5, wherein the gap ratio between the proximal end gap and the distal end gap is 2: 1 to 4: 1 along a longitudinal axis of the cover tile.

7. The construct of any one of claims 1 to 6, wherein the total volume of the wedge-shaped capillary fluid flow gap is 200 to 600 μl.

8. The construct of any one of claims 1 to 7, wherein the longitudinally extended protrusion is a first face, and the lower surface further includes at least one additional face, with the additional face being at an incline relative to the first face.

9. The construct of any one of claims 1 to 7, wherein the longitudinally extended protrusion is a first face, and the lower surface further includes a second face and a third face, wherein the second face and the third face each extend in a lateral direction that is perpendicular to the longitudinal direction of the cover tile and each of the second face and the third face are angled at an incline away from the upper surface of the substrate.

10. The construct of any one of claims 1 to 9, wherein the lower surface of the cover tile and the upper surface of the substrate further define a fluid

accumulator region that is in fluid communication with the wedge-shaped capillary fluid flow gap.

11. The construct of claim 9, wherein at a given cross-sectional position of the cover tile a gap distance between the cover tile lower surface and the upper surface of the substrate at the lateral edge of the cover tile is greater than a gap distance between the longitudinally extended protrusion and the upper surface of the substrate at the same cross-sectional position.

12. The construct of any one of claims 1 to 10, wherein a gap distance between the longitudinally extended protrusion and the upper surface of the substrate at the proximal edge is 300 to 600 μm and a gap distance between the longitudinally extended protrusion and the upper surface of the substrate at the distal edge is 30 to 150 μm.

13. A cover tile for placement over a specimen on a slide, comprising:

wherein the lower surface of the cover tile includes a first face that extends from the fluid inlet port to the fluid outlet port, a second face that extends from the first face to the first lateral edge, and a third face that extends from the first face to the second lateral edge; and wherein the second face and the third face are each tangential to the first face.

14. The cover tile of claim 13, wherein the cover tile has a thickness dimension between the outer surface of the cover tile and the first face that is greater than a thickness dimension between the outer surface of the cover tile and the second face or third face at the first lateral edge or the second lateral edge.

15. The cover tile either claim 13 or 14, wherein the lower surface of the cover tile includes a fourth face located at a region contiguous with the fluid inlet hole and disposed an incline relative to the first face, the second face, and the third face.