Abstract:
A cover ( 10 ) for substrate (not shown) including: a body ( 12 ) defining a cavity ( 18 ), for positioning over the substrate (not shown) to form a reaction chamber ( 18 ); and a projection ( 13 ) extending from the body ( 12 ) to define a fluid reservoir ( 14 ), when the cover ( 10 ) is fitted to the substrate (not shown), the fluid reservoir ( 14 ) being in fluid communication with the cavity ( 18 ).

Description:
FIELD OF THE INVENTION  
       [0001]     The present invention relates to a cover for a substrate, and in one form a cover for use with a microscope slide.  
       BACKGROUND OF THE INVENTION  
       [0002]     Microscope slides are commonly used to view samples of material under a microscope. The samples may contain human tissue, and may require treatment such as staining, so that properties of the sample can be identified. Other materials such as DNA, RNA, or proteins may be included on the slide.  
         [0003]     It is common for several reactions to be undertaken on a sample on a slide. Once the reactions have taken place the slide may be viewed under a microscope. Performing the reactions on the slide can be difficult to automate, as the tissue samples require careful preparation and certain reactions require carefully controlled environments.  
       SUMMARY OF THE PRESENT INVENTION  
       [0004]     In accordance with the present invention, there is provided a cover for a substrate including: 
    a body defining a cavity, for positioning over the substrate to form a reaction chamber; and     a projection extending from the body to define a fluid reservoir, when the cover is fitted to the substrate, the fluid reservoir being in fluid communication with the cavity.    
 
         [0007]     Preferably the cavity extends the full width of a sample holding region on the substrate.  
         [0008]     Preferably, a protrusion extends from the projection, to assist in wicking fluid into the reservoir.  
         [0009]     Preferably, the reservoir is defined between the projection, which is spaced from the substrate, and legs located at sides edge of the cover.  
         [0010]     In one form the projection is formed front two sections, the first section is angled at least at substantially 60° relative to the cavity and the second section is angled at least at substantially 15°.  
         [0011]     In one form, the cover further includes a second reservoir, at an opposite end of the cover.  
         [0012]     Preferably wall portions are located at the edge of the cover, surrounding the cavity on two or more sides.  
         [0013]     In one form the legs extend along the sides of the cover to form the wall portions.  
         [0014]     In a preferred form, the cover includes a locator for controlling and locating the cover, the locator being arranged at an end of the cover opposite the projection.  
         [0015]     In one form the cavity extends to an end edge of the cover adjacent the locator.  
         [0016]     In one form the cover is supported on the substrate by the wall portions.  
         [0017]     Preferably, the cover is made from a polymer material.  
         [0018]     In one form the cavity includes a coating of reduced surface roughness than the polymer material.  
         [0019]     In another form the cavity includes a coating with reduced porosity.  
         [0020]     In another form the cavity has one or more coatings.  
         [0021]     Preferably a first coating is a material having similar properties to the material of the slide.  
         [0022]     Preferably the first coating is silicon dioxide.  
         [0023]     Preferably a second coating is placed intermediate a first coating to provide improved contact properties between the cover and first coating.  
         [0024]     Preferably, the cover has associated wing structures that allow the cover to be engaged and pivoted relative to the substrate so as to open the reaction chamber and allow the slide to be cleared of fluid.  
         [0025]     In another aspect, there is provided a combination of a substrate and a cover, as described above, wherein the cavity of the cover is arranged to face the substrate so as to form a reaction chamber.  
         [0026]     In yet another aspect, there is provided a method of treatment of a sample on a sample holding region of a substrate, including locating a cover, as described above, over the substrate, so that the cavity of the cover faces the substrate to form a reaction chamber over the sample holding region, and depositing fluid into the fluid reservoir to allow the fluid to be drawn into the reaction chamber, as required.  
         [0027]     Preferably, the method further includes sliding the cover relative to the substrate to vary a degree of overlap between the cover and the sample holding region, which results in a corresponding variation in the reaction chamber volume.  
         [0028]     Preferably, the method further includes sliding the cover relative to the substrate until wing structures associated with the cover are engaged and lifted relative to the substrate to pivot the cover into an open condition, and allow fluid to drain from the reaction chamber. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0029]     The invention is described, by way of non-limiting example only, with reference to the accompanying drawings, in which:  
         [0030]      FIG. 1  shows an example of a microscope slide;  
         [0031]     FIGS.  2  ( a )-( c ) show top, side and bottom views of a first example of a cover for a slide;  
         [0032]      FIG. 3  shows a perspective view of the cover of  FIG. 2 ;  
         [0033]     FIGS.  4  ( a )-( c ) show farther views of the cover of  FIG. 2  located on the slide of  FIG. 1 ;  
         [0034]      FIG. 5  is a perspective view of the cover and slide arrangement of  FIG. 4 , showing a cutaway section of the cover;  
         [0035]      FIG. 6  shows a schematic cross section of the cover and slide of  FIG. 5 ;  
         [0036]      FIG. 7  shows a perspective view of a tray adapted to locate covers and slides;  
         [0037]     FIGS.  8  ( a ) and ( b ) show schematic top and sectional side views, respectively, of a further example of a cover;  
         [0038]     FIGS.  9  ( a ) and ( b ) show schematic top and sectional side views, respectively, of a further example of a cover;  
         [0039]     FIGS.  10  ( a ) and ( b ) show schematic top and sectional side views, respectively, of a further example of a cover;  
         [0040]     FIGS.  11  ( a ) and ( b ) show schematic top and sectional side views, respectively, of a further example of a cover,  
         [0041]     FIGS.  12  ( a ) and ( b ) show schematic top and sectional side views, respectively, of a further example of a cover;  
         [0042]     FIGS.  13  ( a ) and ( b ) shows schematic top and sectional side views, respectively, of a further example of a cover;  
         [0043]     FIGS.  14  ( a ) and ( b ) show top and bottom perspective views, respectively, of a further example of a cover;  
         [0044]      FIG. 15  shows a schematic side view of a nose portion of a cover;  
         [0045]      FIG. 16  ( a ) and ( b ) show schematic top and sectional side views, respectively, of a further example of a cover;  
         [0046]      FIG. 17  shows a schematic side view of a further example of a nose portion of a cover;  
         [0047]      FIG. 18  shows the cover of  FIG. 2  mounted to the tray of  FIG. 7 ;  
         [0048]     FIGS.  19  ( a )-( c ) shows the cover of  FIG. 2  in various positions over the slide of  FIG. 1 ; and  
         [0049]     FIGS.  20  ( a ) and ( b ) show a bottom perspective view and enlarged partial perspective view, respectively of a modified cover. 
     
    
     DETAILED DESCRIPTION  
       [0050]     A microscope slide  1  is shown in  FIG. 1  as including an upper surface  2  containing a sample  3 . The slide  1  is identified by a unique bar code  4 . The sample  3 , such as a thinly sliced tissue section, is located on the slide  1  in a sample holding region  5 , which needs to be covered by a cover, such as shown in  FIG. 2 , for subsequent application of test fluids and the like.  
         [0051]     FIGS.  2  ( a )-( c ) and  FIG. 3  show a cover  10  as having a body  12 , a fluid receiving zone  14 , a locating means  16  and a cavity  18  on an underside face  19 . Surrounding the cavity  18  on two sides is a wall portion  20 . At one end of the cover  10 , the wall portion  20  joins with legs  21  which extend upwardly and away from the face  19 . The legs  21  are spanned by a projection  13  which defines a fluid reservoir  17 , between an underside of the projection and the legs  21 .  
         [0052]     The cover  10  is shown fitted to a slide  1  in  FIGS. 4 and 5 . The fluid reservoir  17  is shown roost clearly in  FIG. 4  ( c ) where a detailed view of part of a section A-A taken across the cover  10  and slide  1  is illustrated. The projection  13 , with leg  21  at either end, is raised relative to the slide  1 , to form a volume capable of holding fluid dispensed onto the slide  1 . In this way fluid reservoir  17  enables fluid dispensed onto slide  1  to be held until required, without spilling off an edge of the slide. The projection  13  further assists in spreading the fluid across the full width of the cavity  
         [0053]     The overlap of the cavity  18  with the slide  1  forms what may be described as a reaction chamber, as illustrated in  FIG. 6 . The cavity may vary according to application, typically from 20-200 microns. The wall portion  20  is adapted to support the cover on the slide  1 . The cavityed face  22 , wall portion  20  and sample holding region  5  of a slide  1  form a reaction chamber  24  when the cover  10  is placed at least partially over the sample holding region  5 .  
         [0054]     The fluid reservoir  17  is typically sized to be larger than the volume of the reaction chamber  24 , for example 150% of the volume of the reaction chamber. This provides sufficient volume of fluid to fill the reaction chamber completely, while allowing some excess to flush the chamber, and an amount to be retained in the fluid reservoir to provide a reservoir for evaporation.  
         [0055]     Clamping forces may also be applied to the cover once loaded onto the slide, and these forces are designed to provide a seal between the wall portions  20  and the upper surface of the slide  1 . This is to restrict fluid leakage from the side of the cover. In one example (not shown) the wall portions may have an additional member to assist sealing of the wall portions with the upper surface  2  of the slide  1 . This additional member may be a softer polymer or rubber material.  
         [0056]     The cover  10  also includes engaging surfaces in the form of wings  26 . The wings  26  are adapted to engage ramps  28  of a tray  21  shown in  FIG. 7 , to thereby lift the cover clear of the surface of the slide  1 . An example of the wings lifting the cover free is shown more clearly in  FIG. 18 . The cover  10  may be controlled by an arm (not shown) moving the locating means  16 . The cover  10  may be placed in a number of positions over the slide, exemplified by the positions of the cover relative to the slide shown in  FIG. 19 . In  FIG. 19  ( a ), the cover  10  is in an open position relative to the slide  1 , as the sample is exposed and open.  FIG. 19  ( b ) shows the cover in a partially closed position, and  FIG. 19  ( c ) shows the cover in a fully closed position, where the sample is completely covered by the cover and is therefore wholly contained within the reaction chamber  24 . The reaction chamber formed by the cover and cavity  18 , as shown in  FIG. 5 , extends over most of the slide  1 . However it is possible that the sample may be placed more towards the end of the slide distal from the bar code  4 , and therefore a smaller reaction chamber  24  is required. Reducing the size of the reaction chamber  24  reduces the amount of fluid required to fill the chamber, which can be important where expensive or scarce fluids are used. It is possible to form a smaller reaction chamber with the cover  10 , by only covering a porion of the slide  1  with the cover  10 . This position is shown in  FIG. 18  ( b ).  
         [0057]     Variations in cover constructions are schematically shown in  FIGS. 8-17 . In  FIGS. 8-17 , only the front segments of the covers are shown, and the locating means  16  have been omitted from view for clarity and like parts are denoted by like reference numerals.  
         [0058]     In  FIG. 8  ( a ) a cover  10  is shown having a body  12 , projecting legs  21 , a protruding section  13  and an indent  30 . The projecting legs  21  either side of the body  12  form a fluid receiving zone  14 . When placed onto a slide, fluid may be dispensed into the fluid receiving zone, where it spreads in a circular fashion to contact the protruding section  13 . The indent  30  allows the fluid to contact a wider portion of the protruding section  13  than if the front edge of the protruding section was straight (as shown in  FIG. 9 ). Once the fluid is in contact with the protruding section  13 , it wicks across the width of the cavity  18 . If suction is applied at the rear of the cavity, or the cover is moved along the slide from an open position to a more closed position, then the fluid begins to fill the cavity  18 . When the cavity  18  has moved across the sample  3 , it forms the reaction chamber  24  as the fluid may react with the sample  3 .  
         [0059]     FIGS.  9  ( a ) and ( b ) show a more simple construction of a cover  10  that may be used in some circumstances. The operation of the cover  10  is the same as the operation of the cover  10  in FIGS.  8  ( a ) and ( b ).  
         [0060]      FIG. 10   s  ( a ) and ( b ) show a cover  10  having a body  12  with projecting legs  21 . A protruding section  13  and a bar  31  surround a fluid receiving zone  14  for receiving fluid. The fluid may be dispensed onto the protruding section  13 , where it flows down and onto the slide surface  2 . The protrusion  13  and bar  31  cause the fluid to spread across the width of the cavity  18 , enabling the cavity to be filled with fluid.  
         [0061]     The covers  10  of  FIGS. 11, 12  and  16  operate in similar ways to those described above.  
         [0062]     In relation to all of the above-described coves, it should be appreciated that the covers are generally 25 mm across, and the cavity  18  is typically only 20-200 micrometres high. As such, overall fluid dispense volumes may be in the order of 20-300 microlitres.  
         [0063]      FIG. 13  ( a ) shows another cover  10  having a body  12 , legs  21  and a fluid dispenser  100  dispensing fluid  102  onto the slide  1 . In  FIG. 13  ( a ), the fluid  102  has already been dispensed, and has formed a fluid reservoir in the fluid reservoir  17 . The schematic Figure shows a typical wicking pattern formed by the fluid as it contacts the cover  1 . In  FIG. 13  ( b ), the fluid is just being dispensed onto the projection  13 . In the volumes dispensed, the fluid forms a pool of comparable size to some of the cover features. Not only does the fluid flow forward of the cover as shown in  FIG. 13  ( a ), but it also flows under the cover to at least partially fill cavity  18 . As mentioned above the fluid may be drawn into the cavity further by movement of the cover over the slide or suction applied to the rear of the cavity  18 .  
         [0064]      FIG. 14  ( a ) and ( b ) shows a further embodiment of a cover  10  where like reference numerals are again used to denote like ports. The cover has a fluid reservoir  17 , a projection  13 , and a protrusion in the form of nib  15 . Fluid may be deposited directly On the nib  15  so that the fluid rolls over the projection  13  into reservoir  14 , and to the cavity  18 , as required. If fluid is placed too far ahead of the cover, there are circumstances that may cause the fluid to reach the edge of the slide before wicking across the width of the cavity  18 . It has been found that using the projection  13  causes the dispensed fluid to contact the covertile and spread along the full width of the cavity  13 , due to the positive attraction of the covertile and the fluid. The capillary forces in the cavity cause the fluid to spread out, and the reservoir holds sufficient fluid to ensure that fluid dispensed onto the slide at least fills the cavity  18 . The nib  15  is useful in that if the pipette is not placed to dispense the fluid accurately onto the slide, and for example misses a few millimetres in front of the projection, the nib  15  will be likely to contact the fluid, which will be drawn to the protrusion and into the reservoir. This assists in reducing bubble or void formation within the cavity. The nib  15  may extend approximately 1-5 mm from the projection  13 .  
         [0065]      FIG. 17  shows an example of how fluid spreads across a slide when deposited in front of a cover  50 . A variety of profiles for the underside of a projection  15  may be employed.  
         [0066]     In use, a cover  10  is placed on a slide  1 , as shown in  FIGS. 4, 5  and  6  to cover the sample  3 . The slide  1  will typically be in a tray  21  as shown in  FIG. 7 , said tray  21  able to hold, for example,  10  slides and covers of the examples shown. The tray  21  may then be placed into a biological reaction apparatus, such as that disclosed in Australian Provisional Patent Application No. PS3114/02 by the same applicant, titled “Method and Apparatus for Providing a Reaction Chamber”, filed 20 Jun. 2002, and its associated international patent application, filed 20 Jun. 2003, the contents of which are hereby incorporated by reference.  
         [0067]     Once the tray  21  is loaded into the apparatus (not shown) the slides  1  are held in position, typically at an angle of 5 degrees to the horizontal as shown schematically in  FIG. 13  ( b ),  15  or  17 . The cover  10  is then moved by an arm (not shown) engaging the locating means  16 . Typically, during a sequence referred to as an “open fill”, the cover  10  is moved longitudinally along the surface of the slide  1  until the sample  3  is exposed. A fluid is then dispensed by a dispensing means  100  such as a probe attached to a pump, onto the fluid receiving zone  13  (as shown in  FIG. 13  ( b )). The amount of fluid dispensed is typically sufficient to fill the reaction chamber  24 . The use of the cover  10  with this fill mechanism or methodology allows a small volume of fluid to be uniformly distributed across the reaction chamber  24 . Distributing the fluid across the reaction chamber  24  evenly and without bubbles or air spaces allows reactions to take place on the sample  3  with greater consistency. Also, dispensing fluid into an empty receiving zone where the reaction chamber already contains fluid causes the fluid within the chamber to be replaced by the fluid in the receiving zone minimising mixing of the fluid in the reaction chamber and newly dispensed fluid. The dimensions of the reaction provide a smooth flow of fluid from the reaction chamber such that there is little mixing of the fluids. This is advantageous as it allows a previous fluid to be replaced accurately, with minimal original fluid remaining to contaminate later fluids or reactions. This reduces the number of washes required to clear the reaction chamber  24   
         [0068]     The volume of fluid in a reaction chamber  24  may be, for example 150 microlitres or less, although volumes may vary depending on the application and the reaction chamber dimensions.  
         [0069]     The reaction chamber  24  is able to retain fluid due to the surface tension of the fluid, unless additional fluid is added to the fluid receiving zone, or suction is applied (typically through reduced air pressure) at the end of the slide opposite the fluid receiving zone. The reaction chamber may be filled as it is formed by the cover  10  being moved along the surface of the slide  1  to cover the sample holding region  52 . Alternatively, the reaction chamber may be filled without the cover being moved relative to the slide, due to the process of capillary wicking of dispensed fluid into the reaction chamber.  
         [0070]     In the present examples the cover may be clamped to the slide when not in motion or retracted for an initial fill. The clamping mechanism (not shown) places force around the edge of, for example, cover  10  adjacent the wall portions  20  to locate the cover  10  with respect to the slide  1  during a reaction.  
         [0071]     During the withdrawal of the cover  10  from the slide  1  it is sometimes desirable to remove the cover from contact with the slide. In order to accomplish this, wings  26  engage the ramps  28  to lift the cover clear of the slide. This causes the cover  10  to lift off the slide  1  to prevent fluid contact between the slide  1  and cover  10 . In this way the slide can be cleared of virtually all fluid.  
         [0072]     Parts of the cover may have different material properties compared to the properties of the material of the cover body  12 , which is typically plastic. In one example (not shown) the cavity may have different material properties, in order to provide a reaction chamber  24  with certain material properties. A clear plastic material has been found to be suitable for the body  12  of the cover  10 , to provide suitable mechanical properties such as reasonable strength and rigidity. The cover needs to be sufficiently strong to be moved while clamping forces are applied to the cover, as the clamping forces assist in providing a sealing surface between the walls  20  of the cover  10  and the upper surface of the slide  1 . The cover may be moved to empty or fill the chamber, or also, to promote fluid movement within the reaction chamber to assist a reaction.  
         [0073]     The cover should ideally have some flexibility, as it is desirable that upon application of the clamp, the cavityed face should deflect somewhat. This has been found to assist in moving the fluid within the reaction chamber and therefore increases the exposure of the sample to the fluid.  
         [0074]     Other properties of the cover  10  include the ability to restrict the heat loss from the surface of the slide  1 . Typically the slide will be mounted on a heated block, and the cover will be placed over the sample on the slide. Heating the slide heats the sample and the fluid in the reaction chamber. If there is excessive heat loss from the cover  10  it is difficult to regulate the temperature of the fluid by beating the slide  1 . Further, there may be an excessive temperature gradient across the reaction chamber  24 , which is undesirable.  
         [0075]     The cavityed face  19 , as shown in  FIG. 2 , may have different surface properties to the rest of the cover. It has been found to be desirable to have similar material properties for the upper surface of the slide  2  and the cavity  18 . In one example, it is possible to coat the surface of the cavity with a material, such as silicon dioxide. This coating may be approximately 110 nm thick. The coating provides a surface with material properties similar to that of a glass slide. It has also been found that there are benefits in applying a thin layer (for example 0.5-6 nm) of Chromium Oxide (Cr2O3) to the cavity before applying the silicon dioxide layer. This application of an intermediate layer between the silicon dioxide and plastic provides better adhesion and better thermal expansion properties for the cavity. Further, coatings in general may be used to improve the flatness of the cavity (which reduce nucleation sites and therefore bubble formation at high temperatures). The coatings may be used to modify the capillary flow characteristics of the fluid within the reaction chamber, create an impermeable barrier for gas or liquid between the cover and fluid in the reaction chamber, or provide a chemically inert surface.  
         [0076]     In another example, it is possible to replace the cavityed face  19  with a glass insert supported by the plastic body  12  of the cover  10 . It may also be possible to change the surface properties of the plastic by plasma discharge.  
         [0077]     The covers shown in the examples may be used at temperatures approaching 100 degrees Celsius, especially when used for in-situ hybridisation reactions. At higher temperatures, the fluid evaporates and bubbles are produced. The heating may also cause the cover to bow—the cavity surface is hotter than the top of the cover and expands more, causing the cavity surface to ‘sag’ towards the slide. This helps to remove the bubbles, as the fluid wants to occupy the smaller spaces more than the bubbles do. The bubbles congregate at the ends of the cavity, and must be allowed to escape.  
         [0078]     Experiments have demonstrated that a chamfer at the end of the cavity reliably allows the bubbles to escape to atmosphere. The existing reservoir  17  can be redesigned as illustrated in  FIG. 20 , where a modified cover  60 , similar to that shown in  FIG. 14 , is shown with a chamfer  61  to assist in releasing bubbles, without affecting the even fluid flow through the cavity  18 . The chamfer forms a first angled section  62  at about 60° relative to the cavity and slide surface.  
         [0079]     Fluid evaporation rate is, however, directly linked to the surface area of the fluid exposed to atmosphere—a larger surface area will evaporate faster, and require more frequent replenishment. If the bubble escape angle is steep, the evaporation rate will increase.  
         [0080]     This problem can be overcome by using two angles—a shallow angled section at, say, 15° between the cavity and the chamfer, to minimise evaporation, leading into the steeper angle for bubble release, which also serves to increase the volume of the reservoir.  
         [0081]     The cover  60  is also provided with a second, identically shaped reservoir  63  at an opposite end thereof. The second reservoir  63  can also be used to replenish fluid within the cavity during heating and to allow bubbles to escape. The second reservoir  63  thereby allows for increased control of fluid conditions within the reaction chamber.  
         [0082]     The embodiments of  FIGS. 14 and 20  are considered to represent what is currently believed to be the best known method of performing the cover aspect of the invention.