Abstract:
Disclosed is a cell culture microscopy slide comprising an optically transparent generally flat supporting surface ( 20 ) including upper and lower opposed substrate surfaces ( 27,28 ). A peripheral frame ( 40 ) surrounds the substrate ( 20 ), the frame ( 40 ) having a lower frame surface ( 44 ) and an upper frame surface ( 42 ). The lower frame surface ( 44 ) and the lower substrate surface ( 28 ) are generally flush. The upper frame surface ( 42 ) lies above the upper substrate surface ( 27 ), to form a well ( 32 ), and the upper and lower frame surfaces ( 42,44 ) are continuously flat and generally parallel. The substrate is preferably glass having a thickness of 1.7 mm.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to cell culture microscopy slides and in particular but not exclusively to cell culture slides for imaging in the field of immunoassays, especially immunocytochemistry (ICC). 
       BACKGROUND OF THE INVENTION 
       [0002]    It is known to grow cell cultures on a microscope slide for immunoassay purposes. Generally, the user grows cells on a glass slip by placing the glass into the bottom of a disposable dish. Depending on the cell type, the glass slip may or may not be coated with a surface treatment to encourage cell adhesion to the glass surface, for example, polylysine. The size of slip used typically ranges from 12 mm circular to 25 mm square to 25×75 mm square. At a predetermined time the user removes the slip from the disposable dish with a pair of forceps, hopefully without breaking the delicate glass. The slip is placed into a fixative, normally formaldehyde in a phosphate buffer solution (PBS) but may also be methanol. This chemically fixes expressed proteins in place by cross-linking proteins together. The slip is removed from the fixative and rinsed in PBS. The user then begins a process of adding primary antibodies to the cover glass, rinsing, adding secondary antibodies, rinsing, adding DAPI (4′,6-diamidino-2-phenylindole nuclear stain), rinsing, then adding a small amount of commercially available mounting media, and affixing the slip to a further glass slide with either a sealant, typically fingernail polish, or by using a hardening mounting media. The sealant or mounting media is permitted to harden and the slide assembly is ready for imaging with the stain-labelled proteins sandwiched between two pieces of glass. It is possible, with care, to repeat the steps above with different slips, thereby mounting two or more slips onto a single glass slide. 
         [0003]    The user chooses a magnification for examining the assembled slide. If the objective lens requires a liquid immersion media, the immersion media must be added prior to examining with that objective lens. Some users may use a low magnification, air immersion lens to coarsely determine the focal plane and identify regions of interest to image at higher magnification. The assembled slide is then removed, the objective lens is changed, appropriate immersion liquid is added to either the slide or the objective, the slide is placed back onto the microscope, and the microscope is re-focused onto the sample. 
         [0004]    This method is fraught with problems. In fact, the most difficult part of microscopy is the sample preparation and few users fully comprehend the effect that the sample plays in the optical results. Below are some of the problems faced by users new to immunofluorescence. 
         [0005]    1. Handling the Glass Slip: 
         [0006]    Since the glass slip is delicate and brittle, it is easy to mishandle it. This can result in the glass flipping over during handling. Since the cells are only on one side of the glass and the cells cannot be seen by eye, this leads to preparing the wrong side of the glass wasting expensive or rare materials. Also, the glass is covered with cells which are easily damaged with the forceps needed to handle them. A common problem is that the glass is broken during one of the many preparation steps leading to oddly shaped samples, wasted samples and materials, and sample contamination. Additionally, excessive handling of the glass can result in misidentification of samples which may might necessitate repeating an experiment or worse yet, incorrect results. 
         [0007]    2. Mounting the Glass Slip: 
         [0008]    Since the placement, size, and distribution of the glass slip on the slide is substantially random, there is no option of automatically scanning the slide to find the mounted slip, without scanning the entire slide. Also, since there can be sealant on the both sides of the glass slip, and the sealant can be of varying thickness depending on a number of factors, then the option of automatically moving from one mounted glass slip to another is limited because there is will height differences. There is often more than one glass slip mounted on a slide. Since the addition of mounting media and mounting the cover glass is done manually, these slips are usually in different focal planes which then require that the user refocuses on each glass slip region. Since the biggest microscopy problem is finding the focal plane, this leads to frustration and broken slides. 
         [0009]    3. Material Selection: 
         [0010]    One of the most common problems in ICC is the selection of materials. High numerical aperture lenses are specifically designed for a single glass thickness. There are a myriad of mounting media choices and many of the available choices result in poor imaging. Everything between the sample (e.g. cell) and the detector (e.g. camera) affects the contrast and resolution that can be obtained from the sample. The wrong glass thickness leads to spherical aberration and axial chromatic aberration. The wrong mounting media leads to a number of aberrations, high background, and poor contrast. 
         [0011]    4. Microscopy, Scene Selection, and Sample Bias: 
         [0012]    One of the hardest parts of using the microscope is finding the focal plane. This is especially difficult for sparse samples but can be problematic even for dense samples. Even the “expert” microscopist will occasionally mount the slide upside-down and spend long periods of time trying to find the focal plane only to realize the problem later. A very insidious problem that almost all users are guilty of is selection bias. Microscopists typically choose what cells to image based on visual inspection of the cells. This leads to user bias based on the definition of a “good cell”. This bias is subliminal and difficult to avoid. Furthermore, it makes it difficult for others to repeat an experiment because the “good cell” is subjective. Even if the images being collected are sent to unambiguous image analysis, the selection bias is present. The scientist needs the scene or cell selection to be unbiased as well but this is rarely the case. This leads to dubious results that make their way into questionable papers that others cannot replicate. 
         [0013]    U.S. Pat. No. 7,919,319 discloses culturing cells between two transparent plates spaced by a distance comparable to the size of the cell to be cultured thus providing a monolayer of cells, which can be observed over time by means of microscopy. This technique provides a distorted environment for cell culture because most cells grow best in clumps rather than flat arrays, and cell waste products are less able to be washed away. The technique described is of little use in ICC, because the cells are not intended to be disturbed, and so the washing and staining steps mentioned above are problematic. 
         [0014]    The inventors have devised a microscopy slide which addresses the problems mentioned above. The invention provides a microscopy slide according to claim  1  having preferred features defined by claims dependent on claim  1 . The invention provides also a method as defined by claim  6  and claims dependent thereon. 
         [0015]    The invention extends to any combination of features disclosed herein, whether or not such a combination is mentioned explicitly herein. Further, where two or more features are mentioned in combination, it is intended that such features may be claimed separately without extending the scope of the invention. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0016]    The invention can be put into effect in numerous ways, one illustrative embodiment of which is described below with reference to the drawings, wherein: 
           [0017]      FIG. 1  shows a plan view of a microscopy slide; 
           [0018]      FIG. 2  shows a sectional view of the slide in use along line II-II in  FIG. 1 ; 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0019]    The invention, together with its objects and the advantages thereof, may be understood better by reference to the following description taken in conjunction with the accompanying drawings, in which like reference numerals identify like elements in the Figures. 
         [0020]    Referring to  FIGS. 1 and 2 , a microscope slide  10  is shown which includes an optically transparent generally flat substrate  20  in this case a borosilicate glass, the substrate having opposed upper and lower surfaces  27  and  28 , and an outer plastics moulded peripheral frame  40 . The outer size of the frame conforms to ANSI/SBS 1-2004 and 2-2004 standards. The substrate has a generally uniform thickness of 0.165 mm to 0.175 mm, preferably 0.17 mm. The substrate contains cell culture regions  22  in which cells are grown and staining is performed according to known techniques. In this embodiment, two 13 mm diameter circular regions are illustrated, although just one region could be utilised, or more regions could be used, for example up to 30,000 micro-regions could be used. The regions are separated from each other with hydrophobic material  24  applied to the substrate in all areas except the regions  22 . For micro-regions the hydrophobic material can be applied by a dot matrix printer or similar technique. The hydrophobic material  24  serves to isolate the wells from each other because water-based liquids will avoid the hydrophobic areas  24  and cells will not cross hydrophobic regions. They culture regions  22  are optionally coated with α-polylysine or other commercially available materials which allow cells to adhere to the glass. Also located on the substrate is a unique identifier  26 . In this case the identifier is a bar code strip, which can be read by optical means, including by a microscope to which the slide  10  is mounted. The substrate further includes alignment indicia  48 , in this embodiment located at one corner of the frame, and formed by three raised dimples. The indicia allow the correct orientation of the slide  10  with respect to a microscope, and can be read by the microscope if required. 
         [0021]    The outer frame  40  holds the substrate  20  in place, and has continuously flat upper and lower surfaces  42  and  44 . Herein, ‘continuously flat surfaces’ means surfaces which define an unbroken circuit in one plane around the substrate. The surfaces  42  and  44  are generally parallel to each other. The lower surface  44  includes a recessed window  46  into which snugly fits the glass substrate  20  such that a lower surface  28  of the substrate lies flush with the lower surface  44  of the frame. The upper surface  42  of the frame is higher than the upper surface  27  of the substrate  20 , providing a well  32  in which cell culture media is contained in initial use. 
         [0022]    In use, media, containing cells, is placed into one or both regions  22 , a lid (not shown) is placed over the slide  10 , and the slide is placed into an incubator (not shown). After a period of hours, the cells will settle onto the substrate  20  in the region, spread, and attach to the substrate surface. For longer term cultures, the media containing cells can be removed after about eight hours and the well  32  can be flooded with additional media. The cells will avoid the hydrophobic material  24  and thus no mixing of cells will occur between the wells even when the well  32  is flooded. This technique greatly simplifies the growing of cells, the fixation, the washing, and the entire ICC protocol. 
         [0023]    When the user is ready to proceed with ICC, the media is removed from the region  22  or from the well  32  and approximately 100 μl (for a 13 mm region) of a commercially available fixative is added to each region  22 . Further conventional ICC wash, incubation, wash, incubation, wash, steps are carried out according known techniques but there is no need to handle a delicate glass slip as with conventional techniques. The proteins of interest are stained according to known techniques using labelled antibodies. Upon completion of the ICC steps, approximately 5 μl of conventional mounting media is added to the each region  22  and a sealant slide  50  is placed onto the upper surface  42  of the frame. This protects the regions from dehydration, and contact damage. 
         [0024]    The slide  10  together with the sealant slide  50  (the assembled slide) is placed on a microscope system. Where implemented, the alignment indicia are read to align the microscope table and assembled slide and the bar code identification  26  is read by the system to determine the identity of the slide, and thereby its contents. By cross-referencing the bar code to a list of known slides, the positions of the regions  22  can be readily determined. Additionally, the bar code will include an identifier to uniquely define a particular slide. In this way, there is no ambiguity about the particular sample that is being imaged and removes the need to hand-write identifiers on the slide itself. Immersion oil  52  is applied to the slide and the slide is scanned using a high numerical aperture 4× objective lens  54 , for subsequent higher resolution imaging if required. 
         [0025]    The slide  10  and its use as described above significantly increase the ease of preparation and use of a sample containing slide, particularly for immunofluorescence during ICC experiments where numerous slide preparation steps are needed. The use of the outer plastics frame  40  allows for automated handling of the slide  10  if required, and the frame is constructed to aid automation of the microscope scanning procedure. For example, the slide  10  has a completely flat lower surface  28 / 44 , which allows an object lens (e.g. lens  54 ) to scan that surface without the risk of hitting any protrusions. Using a unique identifier reduces the chances of handling errors, and provides consistent automatic imaging location. 
         [0026]    Although one embodiment only has been described and illustrated, it will be apparent to the skilled addressee that additions, omissions and modifications are possible to those embodiments without departing from the scope of the invention claimed. For example, the preferred substrate  20  is glass but other transparent materials could be employed, for example plastics. The frame  40  is preferable formed from plastics, but this includes thermoplastics and thermosetting plastics. Fibre reinforcement is contemplated. The preferred depth of the well  32  is about 2 to 6 mm, and more preferably 3 to 5 mm because this size accepts about the right volume of cell culture media, but shallower or deeper wells could be used to suit different needs. 
         [0027]    A bar code  26  is described, but other identification means could be used, for example an RFID device could be used to automatically write identification data, and other data to the slide in order to record its preparation progress and the results of any imaging subsequently undertaken. 
         [0028]    The slide  10  could be inverted compared with  FIG. 2 , such that it is viewed from above during imaging. In that case, the terms upper, lower etc. should be construed accordingly and are not intended to be limiting as regards orientation of the slide  10  shown in  FIG. 2 .