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WO2009086487A2 - Couvre-objet liquide, procédé et dispositif pour appliquer et retirer de tels couvre-objets - Google Patents

Couvre-objet liquide, procédé et dispositif pour appliquer et retirer de tels couvre-objets Download PDF

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Publication number
WO2009086487A2
WO2009086487A2 PCT/US2008/088378 US2008088378W WO2009086487A2 WO 2009086487 A2 WO2009086487 A2 WO 2009086487A2 US 2008088378 W US2008088378 W US 2008088378W WO 2009086487 A2 WO2009086487 A2 WO 2009086487A2
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Prior art keywords
liquid
coverslip
liquid coverslip
composition
ethanol
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PCT/US2008/088378
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WO2009086487A3 (fr
Inventor
Marc E. Key
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Spring Bioscience Corp
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Spring Bioscience Corp
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Publication of WO2009086487A3 publication Critical patent/WO2009086487A3/fr
Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N1/31—Apparatus therefor
    • G01N1/312—Apparatus therefor for samples mounted on planar substrates
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/34—Microscope slides, e.g. mounting specimens on microscope slides

Definitions

  • the disclosure concerns a liquid coverslip for mounting tissue samples, such as stained hematological, histological, and cytological specimens, on microscope slides, and a method and device for applying and removing a liquid coverslip.
  • Microscopic methods for analyzing cells or tissues mounted on a glass slide require that the specimen be covered with a transparent mounting medium having a refractive index that usually is greater than about 1.2. This allows the microscopist to observe fine cellular detail at high magnification.
  • the specimen is generally covered with a solid glass or plastic coverslip. Once the specimen is properly mounted it can be observed under a microscope. If the specimen is not mounted, the optical characteristics of the resulting image are diminished due to distortion and diffraction, and fine cellular detail is lost. Therefore, specimen mounting is almost a universal procedure for microscopic analysis.
  • Solid coverslip method utilizes a base layer of mounting medium followed by application of a glass, plastic, or film coverslip. Before the mounting medium is applied, the tissue sample must be dehydrated, and then covered with a liquid layer of an organic solvent that is miscible with the mounting medium. Once the solid coverslip is applied, the mounting medium dries, such that the solid coverslip becomes permanent.
  • Solid coverslips can present several disadvantages when attempting to view a sample under a microscope. For example, solid coverslips may trap air underneath, which can cause significant optical distortion of the sample.
  • the second method referred to herein as the liquid coverslip method, involves mounting the specimen with a liquid mounting medium that dries to form a solid coverslip.
  • a liquid mounting medium that dries to form a solid coverslip.
  • the mounting medium used with this method can be water soluble.
  • conventional liquid coverslip compositions and methods of applying them result in a lower optical resolution when the sample is viewed under a microscope. Reduced optical resolution presumably occurs because the refractive indices of liquid coverslips are lower than those of solid coverslips. Additionally, liquid coverslips are more prone to scratches and other damage, because the coverslip produced using a conventional liquid coverslip composition is not as hard as solid coverslips.
  • coverslips typically are applied by placing a few drops of the liquid onto the slide. The drops coalesce and produce a convex pool of liquid on the slide. This can result in a thick and uneven coating on the sample.
  • the coverslip composition dries from edge to center, forming concentric rings. These characteristics may contribute to the lower optical resolution previously mentioned.
  • coverslips typically are permanently applied as the last processing step before microscopic examination.
  • U.S. Patent No. 5,492,837 discloses "compositions and methods for permanently mounting tissue sections on microscope slides using an aqueous solution of polyvinylpyrrolidone.” The '837 Patent, Col. 1, line 65, to Col. 2, line 1 (emphasis added).
  • Disclosed embodiments of a liquid coverslip can have a higher refractive index than conventional liquid coverslips. This is due, at least in part, to the materials used in the composition.
  • Prior art liquid coverslips typically contained glycerol, which could lower their refractive indices.
  • the present liquid coverslips need not contain glycerol, and thus can have an improved refractive index.
  • Liquid coverslips of the present disclosure can contain an alcohol, such as a lower alkyl alcohol, one example being ethanol, and thus have a reduced viscosity when compared to prior art mounting medium.
  • Prior art liquid coverslips often required a higher viscosity for manual application; otherwise the liquid coverslip would not stay in place on the slide.
  • Liquid coverslips disclosed herein can have a lower viscosity than prior coverslips enabled.
  • One reason for this is the automated method of application, also disclosed herein.
  • the present liquid coverslips produce a thin, uniform layer on the specimen, in part because of this lower viscosity.
  • the thin, uniform layer may improve the optical quality of the present liquid coverslip, when compared to prior art liquid coverslips.
  • a method and a device for applying and removing a liquid coverslip so that recycling and sequential staining can be performed are also disclosed.
  • a uniformly thin layer of the present liquid coverslip composition can be formed over the tissue sample by applying the composition to a surface of a microscope slide and spreading the composition over the slide and tissue sample using, for example, a spreader, instead of simply placing drops onto the tissue sample.
  • Application of the present liquid coverslip can also be automated, such as by a robotically controlled mechanical spreader.
  • Removing disclosed embodiments of the liquid coverslips can be easier and quicker than removal of prior art coverslips. Removal can be automated or accomplished manually, and can be accomplished simply by placing the slide in a liquid bath at an elevated temperature for about thirty seconds. The tissue sample is usually not damaged during liquid coverslip removal. Additionally, if removal of immunological components, such as staining, is also desired, these can be removed either sequentially or simultaneously with liquid coverslip removal, such as by placing the slide in a liquid bath at an elevated temperature for a period of time effective to remove any such components, such as for about fifteen minutes.
  • this disclosure describes a method for repairing a scratched or damaged liquid coverslip, and describes a method of placing a solid coverslip over a liquid coverslip. Placing a second layer of liquid coverslip on top of a damaged or scratched liquid coverslip can repair the damage and restore image quality when the slide is viewed under a microscope.
  • FIG. 1 is a perspective view illustrating one method of applying a liquid coverslip to a slide, involving pulling the material across the sample.
  • FIG. 2 is a cross sectional view illustrating a liquid coverslip after application to a microscope slide.
  • FIGS. 3A-3B are enlarged plan views of a tissue section, illustrating a microscopic image before and after repair of a damaged liquid coverslip.
  • Antigen A molecule that stimulates an immune response. Antigens are usually proteins or polysaccharides. An epitope is an antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, such that they elicit a specific immune response. An antibody binds a particular antigenic epitope. The binding of an antibody to a particular antigen or epitope of an antigen can be used to localize the position of the antigen, for example in or on a biological sample, or determine if the particular antigen is present in a biological sample.
  • Antibody A polypeptide ligand that includes at least a light chain or heavy chain immunoglobulin variable region and specifically binds an epitope of an antigen.
  • Antibodies can include monoclonal antibodies, polyclonal antibodies, or fragments of antibodies.
  • an antibody is labeled with a detectable label such as an enzyme or fluorophore.
  • the term "specifically binds" refers to, with respect to an antigen, the preferential association of an antibody or other ligand, in whole or part, with a specific polypeptide, such as a specific double-stranded DNA binding protein, for example a transcription factor, such as an activated transcription factor.
  • a specific binding agent binds substantially only to a defined target. A minor degree of nonspecific interaction may occur between a molecule, such as a specific binding agent, and a non-target polypeptide. Nevertheless, specific binding can be distinguished as mediated through specific recognition of the antigen. Although selectively reactive antibodies bind antigen, they can do so with low affinity.
  • Specific binding typically results in greater than 2-fold, such as greater than 5 -fold, greater than 10-fold, or greater than 100-fold increase in amount of bound antibody or other ligand (per unit time) to a target polypeptide, such as compared to a non-target polypeptide.
  • a variety of immunoassay formats are appropriate for selecting antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
  • Antibodies are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody.
  • VH region and VL region are responsible for binding the antigen recognized by the antibody.
  • a scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains.
  • the term also includes recombinant forms such as chimeric antibodies (for example, humanized murine antibodies) and heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, Immunology, 3rd Ed., W. H. Freeman & Co., New York, 1997.
  • a “monoclonal antibody” is an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected.
  • Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody- forming cells from a fusion of myeloma cells with immune spleen cells. These fused cells and their progeny are termed "hybridomas.”
  • Monoclonal antibodies include humanized monoclonal antibodies.
  • Placement in direct physical association for example both in solid form and/or in liquid form (for example, the placement of a biological sample in contact with liquid coverslip composition).
  • Fixation A process which preserves cells and tissue constituents in as close to a life-like state as possible and allows them to undergo preparative procedures without change. Fixation arrests the autolysis and bacterial decomposition processes which begin upon cell death, and stabilizes the cellular and tissue constituents so that they withstand the subsequent stages of tissue processing, such as for IHC or ISH. Tissues may be fixed by either perfusion with or submersion in a fixative, such as an aldehyde (such as formaldehyde, paraformaldehyde, glutaraldehyde, and the like).
  • a fixative such as an aldehyde (such as formaldehyde, paraformaldehyde, glutaraldehyde, and the like).
  • fixatives include oxidising agents (for example, metallic ions and complexes, such as osmium tetroxide and chromic acid), protein-denaturing agents (for example, acetic acid, methanol, and ethanol), fixatives of unknown mechanism (for example, mercuric chloride, acetone, and picric acid), combination reagents (for example, Carnoy's fixative, methacarn, Bouin's fluid, B5 fixative, Rossman's fluid, and Gendre's fluid), microwaves, and miscellaneous (for example, excluded volume fixation and vapor fixation).
  • Additives may also be included in the fixative, such as buffers, detergents, tannic acid, phenol, metal salts (for example, zinc chloride, zinc sulfate, and lithium salts), and lanthanum.
  • formaldehyde generally in the form of a formalin solution (4% formaldehyde in a buffer solution, referred to as 10% buffered formalin).
  • Fluorophore A chemical compound, which when excited by exposure to a particular stimulus, such as a defined wavelength of light, emits light (fluoresces), for example at a different wavelength (such as a longer wavelength of light). Fluorophores are part of the larger class of luminescent compounds.
  • Luminescent compounds include chemiluminescent molecules, which do not require a particular wavelength of light to luminesce, but rather use a chemical source of energy. Therefore, the use of chemiluminescent molecules (such as aequorin) can eliminate the need for an external source of electromagnetic radiation, such as a laser.
  • fluorophores that can be used in the methods disclosed herein are provided in U.S. Patent No. 5,866,366 to Nazarenko et ah, such as 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'- aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS), 4-amino-N-[3- vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4- anilino-l-naphthyl)maleimide, anthranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7- amino-4-trifluoromethylcoumarin (Coumarin
  • fluorophores include those known to those skilled in the art, for example those available from Invitrogen (Eugene, OR), including without limitation fluorescent nanocrystals (such as QDots®).
  • Immunohistochemistry A method of determining the presence or distribution of an antigen in a sample by detecting interaction of the antigen with a specific binding agent, such as an antibody.
  • Label An agent capable of detection, for example by spectrophotometry, flow cytometry, or microscopy.
  • a label can be attached to a specific binding agent, such as an antibody, thereby permitting detection of the specific binding agent and hence an antigen bound by the specific binding agent.
  • labels include, but are not limited to, radioactive isotopes, enzyme substrates, co- factors, ligands, chemiluminescent agents, fluorophores (such as small molecule fluorophores or semiconductor nanocrystals), haptens, enzymes, and combinations thereof.
  • a sample such as a biological sample, is a sample that includes biological materials (such as nucleic acid and proteins) obtained from an organism or a part thereof, such as a plant, animal, bacteria, and the like.
  • the biological sample is obtained from an animal subject, such as a human subject.
  • a biological sample is any solid or fluid sample obtained from, excreted by or secreted by any living organism, including without limitation, single celled organisms, such as bacteria, yeast, protozoans, and amebas among others, multicellular organisms (such as plants or animals, including samples from a healthy or apparently healthy human subject or a human patient affected by a condition or disease to be diagnosed or investigated, such as cancer).
  • a biological sample can be a biological fluid obtained from, for example, blood, plasma, serum, urine, bile, ascites, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, a transudate, an exudate (for example, fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (for example, a normal joint or a joint affected by disease, such as a rheumatoid arthritis, osteoarthritis, gout or septic arthritis).
  • a biological sample can also be a sample obtained from any organ or tissue (including a biopsy or autopsy specimen, such as a tumor biopsy) or can include a cell (whether a primary cell or cultured cell) or medium conditioned by any cell, tissue or organ.
  • a biological sample is a nuclear extract.
  • a biological sample is bacterial cytoplasm.
  • the present liquid coverslips typically are used with microscope slides having standard compositions and dimensions, which typically are approximately 1 inch by 3 inches. However, all types of slides can be used with the disclosed liquid coverslips.
  • a microscope slide has a label on one end of the slide, approximately one square inch in area. This label can be used to provide identifying information. This portion of the slide is generally not viewed under the microscope. The remaining area is the typical viewing area of a standard microscope slide.
  • the liquid covers lip of the present disclosure also can be used with conventional specimens and procedures.
  • the liquid coverslips can be used to cover tissue samples being examined using immunohistochemistry (IHC) or in situ hybridization (ISH) techniques. Disclosed embodiments of the liquid coverslips are not limited for use with IHC and ISH techniques, but may find particular utility in these fields.
  • the tissue can be fixed and embedded in paraffin, in accordance with standard procedures.
  • a thin tissue slice can then be cut and applied to a microscope slide.
  • Multiple tissue slices can be cut and applied to separate microscope slides in the same manner as above.
  • the tissue sample often is dewaxed. Dewaxing can be accomplished by submerging the sample in a paraffin solvent. Paraffin solvents typically are aromatic solvents, such as xylene, and/or toluene.
  • the tissue sample is then subjected to an alcohol solvent, and finally is subjected to an aqueous solution.
  • Probes and/or antibodies then can be applied to the tissue sample and allowed to react and bind to any target molecules present, such as in conventional staining techniques.
  • Secondary reagents stains and counterstains
  • a liquid coverslip of the present disclosure can be applied to protect the tissue sample.
  • the present liquid coverslips comprise an effective amount of a polymer, an organic solvent, and optional other materials, such as plasticizers, antimicrobials, and/or preservatives, with the balance being water.
  • Certain disclosed embodiments comprise polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), or a combination thereof.
  • PVA polyvinyl alcohol
  • PVP polyvinyl pyrrolidone
  • the organic solvent can be an organic solvent chosen to provide sufficient volatility for the desired speed of drying for the particular application.
  • Vapor pressure is a measure of volatility, or the rate at which a liquid evaporates. Liquids of high vapor pressure evaporate rapidly, while those of low vapor pressure evaporate more slowly.
  • the liquid covers lip of the present disclosure may contain water, having a vapor pressure of 17.3 at 20 0 C, and, for example, ethanol, having a vapor pressure of 43.38 at 20 0 C.
  • the ethanol effectively increases the vapor pressure of the liquid coverslip, making it dry more rapidly than a mounting medium composed primarily of water. Rapid drying of the mounting medium can provide slides that are ready for viewing under the microscope within a few minutes. Rapid drying can also ensure a fixed refractive index.
  • the organic solvent is miscible with water, and typically is a lower alkyl alcohol, such as methanol, ethanol, isopropyl, propanol, or butanol.
  • a lower alkyl alcohol such as methanol, ethanol, isopropyl, propanol, or butanol.
  • Any sufficiently volatile chemical, such as alcohols in addition to lower alkyl alcohols, ethers, etc., can replace or be mixed with a first lower alkyl alcohol, such as ethanol, so long as such additives substantially evaporate while the liquid coverslip is drying and does not substantially change the staining patterns of the tissues being examined, such as by dissolving or reacting with one or more of the sample stains.
  • the vapor pressure of the liquid coverslip composition is greater than about 17.3 to ensure fast drying.
  • additional components such as one or more plasticizers, preservatives and/or antimicrobials also may be present in the liquid coverslip.
  • Any preservative or anti-microbial agent is used in an effective but relatively low amount, typically less than about 2% by volume, more typically about 1% or less, and even more typically such components would only be present at a fraction of a percent.
  • Antimicrobial agents may include bronidox, proclin, benzalkonium chloride, thimerosal, and sodium azide, and combinations of these, as well as any other suitable antimicrobial agent.
  • Plasticizers may include polyisobutylene, polyisoprene, polyethylene, polypropylene, and combinations of these, as well as any other suitable plasticizer.
  • Polymers such as PVP and PVA are available in a number of average molecular weights.
  • the only limits on the average molecular weight for PVA and PVP used in the liquid coverslips are that the polymer be soluble, or at least substantially so, in water to an extent sufficient to allow it to be used as described herein, and that the aqueous solution dries to form a protective and substantially transparent surface.
  • PVA is prepared by partial or complete hydrolysis of polyvinyl acetate to remove acetate groups.
  • PVA is also available in a number of different percents hydrolysis, such as from about 80% hydrolyzed to greater than 99% hydrolyzed.
  • PVA is sold commercially by many different companies, such as Kuraray America Inc. (New York, NY), which sells PVA under the name Mowiol.
  • PVP is available from a number of different companies, including Sigma- Aldrich (St. Louis, MO).
  • Disclosed liquid coverslips can improve the refractive index over prior liquid coverslips.
  • Most prior art mounting medium compositions had a refractive index of frp, about 1.40 to about 1.45 (aqueous-based) or from about 1.45 to about 1.50 (non- aqueous-based).
  • the refractive index of disclosed liquid coverslips is preferably above about 1.33, the refractive index of water.
  • the refractive index of disclosed liquid coverslips is more preferably above about 1.45, the refractive index of glycerol, and can be as high as 1.6 of greater.
  • the refractive index of the liquid coverslip is close to that of the refractive index of the tissue sample (typically from about 1.45 to about 1.55) as well as the microscope slide (about 1.50 for a glass slide).
  • the refractive index of a mounting medium is lower or higher than the refractive index of the tissue sample, the stained cells may appear distorted.
  • the refractive index of the mounting medium of the present disclosure can be substantially equal to the refractive index of the pure polymeric component.
  • the refractive index is approximately 1.53.
  • Disclosed liquid coverslip embodiments comprise the polymer in an amount sufficient to optimize the refractive index of the liquid coverslip, while still allowing the liquid coverslip to dry relatively quickly to form a smooth, even covering over the tissue sample.
  • An appropriate amount for the polymer typically is an amount greater than zero to about 20% by volume, preferably from about 5% by volume to 10% by volume, and even more preferably about 9% by volume.
  • a liquid coverslip comprise a lower alkyl alcohol, such as methanol, ethanol, or isopropanol, present in amount sufficient to optimize the spreading characteristics of the liquid coverslip and lower the surface tension.
  • ethanol was added to the liquid coverslip.
  • the surface tension of ethanol is typically determined to be about 22.1, while water has a surface tension of 72.8.
  • a lower surface tension reduces beading on the surface of the tissue and microscope slide, and thus may help enable a thinner, more uniform coat of the liquid coverslip when compared to coverslips with higher surface tension.
  • the optimal amount of ethanol for disclosed embodiments was determined to be less than about 35% by volume, preferably about 33% by volume, which caused the mounting medium to spread in a uniform layer across the surface of the glass slide, and to dry without forming concentric drying rings.
  • the surface tension of the liquid coverslip is preferably less than about 72.8 (the surface tension of water).
  • One current working embodiment of a disclosed coverslip composition comprised about 9% by volume of PVA, about 33% ethanol, and about 58% water.
  • the PVA used was Mowiol 8-88, having a viscosity of between 7.0 and 9.0 millipascal-seconds (determined with 4% aqueous solution at 20 0 C) and a percent hydrolysis between 86.7% and 88.7%. Mowiol is also available in a number of other viscosities and percents of hydrolysis.
  • the composition was made at an initial concentration of about 13.5% PVA (w/v) in water. After dissolving, the PVA mixture was then further diluted with 100% ethanol at a ratio of two parts of PVA mixture to 1 part of ethanol.
  • the final mounting medium contained 9% (w/v) PVA in 33% ethanol. Ethanol reduces the viscosity of some embodiments of the disclosed liquid coverslip, thus making the composition more amenable to automated application and removal.
  • the liquid coverslip composition was applied to the slide, the ethanol and water were substantially completely evaporated to form the liquid coverslip.
  • the liquid coverslip can be applied manually to a microscope slide by pipetting an appropriate amount, such as about 100 ⁇ L, onto one end of the slide.
  • a spreader can be used to spread the liquid coverslip composition substantially evenly across the viewable surface of the slide, thus providing a thin, uniform layer of liquid coverslip material. The spreader can be designed to maintain a suitable gap above the surface of the microscope slide to avoid damaging the tissue.
  • this gap can be about 0.5 mm. This gap can allow the tissue to pass under the spreader without contact between the two. Therefore, if the size of the tissue sample permits, the gap can be smaller, or the gap can be larger for a thicker tissue sample or if a thicker layer of liquid coverslip is desired.
  • FIG. 1 which is not drawn to scale, illustrates a method of pulling the liquid coverslip composition across a horizontal slide.
  • a microscope slide such as a glass or plastic microscope slide, provides a surface for a tissue sample 2.
  • Liquid coverslip composition 3 is applied to the microscope slide 1 and tissue sample 2, and spread across tissue sample 2 using a spreader 4.
  • Liquid coverslip composition 3 is pulled by spreader 4 using a capillary action in this embodiment.
  • liquid coverslip composition 3 may be pushed by (rather than pulled by) a spreader such as spreader 4 of FIG. 1.
  • Spreader 4 may take any form suitable for spreading the liquid coverslip composition over a microscope slide and tissue sample, and need not look substantially similar to the spreader 4 seen in FIG. 1.
  • suitable spreaders for applying a liquid coverslip composition of the present disclosure may comprise a straight edge, a flat edge, a tapered end, may provide a substantially flat surface for spreading, and/or may comprise a roller mechanism.
  • Spreader 4 may be made of any suitable material for spreading a liquid coverslip composition of the present disclosure.
  • spreader 4 may be made of a metal, plastic, glass, ceramic, wood, fiberglass, fabric, rubber, combinations of these, and/or any other suitable material.
  • Spreader 4 may be an independent device, or it may be integrated into a system which performs multiple functions.
  • spreader 4 may both pipette the liquid coverslip composition onto the slide, as well as spread the liquid coverslip composition over the slide surface and tissue sample surface.
  • FIG. 2 which is not drawn to scale, illustrates a cross-sectional view of liquid coverslip 3 after application to microscope slide 1. Tissue sample 2 is shown on a surface of microscope slide 1. Liquid coverslip 3 has been applied to microscope slide 1 and tissue sample 2, such that liquid coverslip 3 at least substantially envelops tissue sample 2. Liquid coverslip 3 coats and surrounds tissue sample 2, providing a protective coating over at least a portion of the tissue sample 2 and microscope slide 1.
  • Liquid coverslip 3 may be present on substantially all of the viewable portion of the microscope slide 1, as depicted in FIG. 2. In some embodiments, however, liquid coverslip 3 may be present on a lesser portion, and may substantially cover only tissue sample 2. The depiction in FIG. 2 of the shape of liquid coverslip 3 should not be seen as limiting in any way. For example, liquid coverslip 3 appears essentially as a rectangular box on one surface of microscope slide 1, covering tissue sample 2. However, in some embodiments, liquid coverslip 3 may appear to be substantially amorphous, lacking any sharp corners. In other embodiments, liquid coverslip 3 may have a sloped shape, where the thickness of the liquid coverslip 3 gradually increases from the edge of microscope slide 1 to the edge of tissue sample 2.
  • Liquid coverslips compositions of the present disclosure may be applied using a pipetting device to apply the liquid coverslip composition and a spreader manually operated to spread the liquid coverslip composition.
  • the liquid coverslip composition may be applied by an automated robotic mechanism, such as a robotic arm that has both pipetting and mechanical spreading capabilities.
  • the liquid coverslip is allowed to dry at either room temperature, or at an elevated temperature, preferably at a temperature of about 60 0 C or less.
  • the drying temperature primarily is a question of drying time.
  • the liquid coverslip can be dried in a short amount of time, such as in about two minutes, at temperatures greater than ambient. Alternatively, if the liquid coverslip is dried at room temperature, drying can take about twenty minutes. Drying can be done at any temperature, so long as the temperature is lower than the decomposition temperature of the polymer used in the liquid coverslip and does not damage or impair visibility of the tissue sample. The tissue sample is now ready for viewing under a microscope.
  • the liquid coverslip subsequently can be removed without damaging the underlying tissue sample. Because the liquid coverslip is so thin and in part due to the composition, the liquid coverslip of the present disclosure can be easily removed. Removal can comprise only removing the liquid coverslip, or it can also comprise simultaneous removal of both the liquid coverslip and any staining previously applied to the tissue sample. Removing just the liquid coverslip can be accomplished in as little as about thirty seconds by placing the microscope slide in a liquid bath at an elevated temperature.
  • the liquid bath may comprise an inorganic solvent, such as an aqueous solvent, or water. While not meant to be limiting, one example of a preferred bath is a Coplin jar containing enough hot water to immerse the coverslipped portion of the slide.
  • the Coplin jar itself may be placed in a water bath at an elevated temperature in order to maintain the temperature of the Coplin jar and its contents.
  • the liquid bath can comprise any aqueous solution so long as it does not damage the underlying tissue sample after or during removal of the liquid coverslip, such as any aqueous solution or aqueous buffer.
  • One common liquid bath is water.
  • Common buffers used in histology include phosphate buffers, phosphate buffered saline, Tris, acetate and citrate. However, if a user is going to re-stain the tissue with a different stain which required the tissue to be in a certain buffer, the user may choose to use this same buffer to remove the coverslip.
  • Dilute acid solutions also could be used to remove the coverslip.
  • Dilute acid solutions also could be used to remove certain stains, such as hematoxylin. If a user desired to use acid to remove a certain stain, the same acid also might be used to remove the mounting medium, thereby accomplishing both in a single step.
  • Dilute acid solutions also can be used to remove antibodies and stains based on antibodies. However, heat is more efficient at removing antibodies.
  • any staining that is based on antibodies, and/or probes can be accomplished by placing the slide in water at an elevated temperature for a longer period of time. Removal methods vary depending on the type of stain employed. For IHC components, some stains can be removed by submerging the slides in water elevated to about 60 0 C, for about fifteen minutes. The antibodies that are used for these stains begin to release at about 56 0 C, and will be essentially completely eluted after about fifteen minutes. However, even at three minutes, significant amounts of the antibodies will have been eluted.
  • Direct conjugates stains that are directly attached to the antibody, are themselves eluted when the antibody is eluted. Fluorochromes are typical of direct conjugates. Common fluorochome stains include fluorescein (green), rhodamine (red), Texas Red (red). Numerous other colors are also available. These stains generally will be removed from the tissue simultaneously with liquid coverslip removal. In indirect methods, the detectable labels are not directly conjugated to the antibody. Therefore, when the antibody is eluted the labels remain on the tissue. This is typical of most IHC methods that utilize enzymes such as peroxidase or alkaline phosphatase. These stains may remain on the tissues after the liquid coverslip and antibodies have been removed.
  • nucleic acid probes can be removed using water heated to an effective temperature, such as about 100 0 C, for an effective period of time, which may be about fifteen minutes.
  • the liquid coverslip alone can be removed by placement in a bath heated to a temperature of about 56 0 C, and the probe would remain with the tissue sample.
  • a stain such as a fluorochrome.
  • the fluorochrome will be removed along with the probe, just as described above in connection with antibodies.
  • the probe can be part of an indirect method, in which case the stain will remain behind, even if the probe is removed.
  • the tissue sample is typically unharmed by removing the liquid coverslip and/or prior stains. As a result, the tissue sample can be re-stained, re-coverslipped, and reexamined microscopically.
  • the resulting images can be digitally recorded or manually recorded and used as individual images, or digitally combined with the images from the first set of stains.
  • Such digitally combined images can be produced using image processing software such as Adobe Photoshop and displayed on a computer.
  • the processes of applying and removing the present liquid coverslip can be automated, such as by a robotic arm mechanism, or performed manually.
  • an automated device for applying a liquid coverslip can be integrated into a single system which not only applies one or more liquid coverslips, but also can remove the liquid coverslip after a slide has been viewed.
  • An apparatus that applies and removes the liquid coverslip could use the following steps: 1. The coverslips are removed by laying the slides horizontally onto a heating platform. A dispensing device dispenses water onto the slides, and the slides are heated to dissolve the coverslip. 2. After the coverslips are dissolved the slides may be removed from the device for re-staining, or the device may also contain a staining component.
  • the slides are re- stained.
  • an automated dispensing device dispenses the liquid coverslip onto the slides, and an automated spreader spreads the liquid coverslip.
  • the heating platform then heats to about 60 0 C to complete the drying process.
  • FIGS. 3A-3B illustrate an example of a repaired liquid coverslip.
  • FIG. 3A illustrates an enlarged plan view of a tissue sample covered with a liquid coverslip, as seen under a microscope.
  • the liquid coverslip covering the sample of FIG. 3 A includes a scratch 10.
  • a scratch in the liquid coverslip can impair the image quality of the microscopic image, as seen in FIG. 3 A.
  • the liquid coverslip can be repaired by applying another liquid coverslip layer, and the results can be seen in FIG. 3B. As seen in FIG. 3B, the optical clarity of the liquid coverslip has been restored. A harder or more permanent outer layer may be desired for certain applications.
  • the liquid coverslip of the present disclosure can be overlaid with a solid coverslip, such as a glass, plastic, or film coverslip.
  • the present liquid coverslip can be used with any method of preparing a tissue for microscopic examination, not just the specific methods described above.
  • other methods of preparing tissue for microscopic examination include applying different fixatives, such as other formaldehyde-based fixatives or alcohol- based fixatives, or embedding the tissue into gelatin, agarose, or into a frozen block for sectioning rather than a paraffin block.
  • Plasticizers also may be used with the paraffin or other fixative, in order to aid in tissue sectioning.
  • IHC staining and ISH staining there are numerous alternative methods for performing tissue pre-treatment. Any of these methods would be compatible with the present liquid coverslips. In fact, any stain used in pathology or histology would be compatible with the present invention.
  • disclosed embodiments of the present liquid coverslip are not limited to the microscopic imaging methods mentioned above. Any microscopic method currently known or subsequently developed will be compatible with the present liquid coverslips. Such alternative microscopic methods include computer- aided image analysis of digital images.

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Abstract

L'invention concerne des compositions de couvre-objets liquides pouvant être appliquées sur un porte-objet de microscope au moyen d'un système automatisé. L'invention concerne également un dispositif et un procédé pour appliquer de telles compositions. Les couvre-objets liquides permettent un retrait automatisé de ceux-ci, ainsi qu'une réparation facile des couvre-objets endommagés ou égratignés. L'invention concerne encore des dispositifs et des procédés pour retirer et réparer des couvre-objets de manière automatisée. Une telle application et un tel retrait automatisés permettent de recycler du tissu de manière plus pratique. Les compositions de couvre-objets liquides peuvent comprendre un polymère, notamment un alcool polyvinylque ou un polyvinylpyrrolidone, ainsi que de l'éthanol et de l'eau. Les couvre-objets liquides peuvent également contenir des plastifiants, des agents antimicrobiens et/ou des conservateurs. Ces couvre-objets liquides permettent d'éviter d'avoir recours à des couvre-objets solides ou en plastique.
PCT/US2008/088378 2007-12-28 2008-12-26 Couvre-objet liquide, procédé et dispositif pour appliquer et retirer de tels couvre-objets Ceased WO2009086487A2 (fr)

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WO2013155064A1 (fr) * 2012-04-10 2013-10-17 Rutgers, The State University Of New Jersey Agent de clarification et milieu de montage pour microscopie
WO2016123868A1 (fr) * 2015-02-06 2016-08-11 丁伟 Procédé de préparation de produit de contrôle de qualité pathologique de chute ou de revêtement à l'état liquide, et ses utilisations
CN107817143A (zh) * 2017-10-20 2018-03-20 杭州依美洛克医学科技有限公司 用于免疫组化物检测配合显微镜载物片的生物盖片
JPWO2016203952A1 (ja) * 2015-06-16 2018-04-05 コニカミノルタ株式会社 病理標本、病理標本の作製方法、および蛍光画像の取得方法
US10018540B2 (en) 2012-04-10 2018-07-10 Rutgers, The State University Of New Jersey Clearing agent and mounting media for microscopy
CN111247415A (zh) * 2017-09-11 2020-06-05 生命科技股份有限公司 折射率匹配配制物
US12234505B2 (en) 2010-04-05 2025-02-25 Prognosys Biosciences, Inc. Spatially encoded biological assays
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US6939032B2 (en) * 2001-10-25 2005-09-06 Erie Scientific Company Cover slip mixing apparatus
KR100494979B1 (ko) * 2003-07-14 2005-06-14 주식회사 정우인터내셔날 조직 절편 보호 및 해상력 증진용 액상 커버 슬립, 그제조용 조성물, 그로부터 제조된 커버슬립을 구비한슬라이드 구조체 및 그 제조방법

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US11519863B2 (en) 2012-02-21 2022-12-06 Leica Biosystems Nussloch Gmbh Apparatus for checking the coverslipping quality of samples for microscopic examination
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US20130217065A1 (en) * 2012-02-21 2013-08-22 Leica Biosystems Nussloch Gmbh Method in the preparation of samples for microscopic examination, and apparatus for checking the coverslipping quality of samples
US9880079B2 (en) * 2012-02-21 2018-01-30 Leica Biosystems Nussloch Gmbh Method in the preparation of samples for microscopic examination and for checking coverslipping quality
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US9464971B2 (en) 2012-04-10 2016-10-11 Rutgers, The State University Of New Jersey Clearing agent and mounting medium for microscopy
US10067041B2 (en) 2012-04-10 2018-09-04 Rutgers, The State University Of New Jersey Clearing agent and mounting medium for microscopy
US10663377B2 (en) 2012-04-10 2020-05-26 Rutgers, The State University Of New Jersey Clearing agent and mounting media for microscopy
US10018540B2 (en) 2012-04-10 2018-07-10 Rutgers, The State University Of New Jersey Clearing agent and mounting media for microscopy
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US10359345B2 (en) 2015-02-06 2019-07-23 Wei Ding Method for preparing liquid-state dripping or coating pathological quality control product and uses thereof
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JPWO2016203952A1 (ja) * 2015-06-16 2018-04-05 コニカミノルタ株式会社 病理標本、病理標本の作製方法、および蛍光画像の取得方法
CN111247415A (zh) * 2017-09-11 2020-06-05 生命科技股份有限公司 折射率匹配配制物
US11988585B2 (en) 2017-09-11 2024-05-21 Life Technologies Corporation Refractive index matching formulations
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US12365935B2 (en) * 2021-05-06 2025-07-22 10X Genomics, Inc. Methods for increasing resolution of spatial analysis

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