Patent Application
20230280579
The invention relates to microscope slides, in particular microscope slides for histological or cytological use, for example with a non-inverted, bright field optical microscope, comprising a histological or cytological sample in contact with an optically transparent coverslip or base, wherein the optical microscope can be used to study the sample through the optically transparent coverslip or base.
Microscope slides for holding specimens, such as tissue samples, have been available for over two hundred years.
Typically microscope slides are a thin flat piece of glass, typically 75×25 mm and approximately 1 mm thick, used to hold objects for examination under the optical microscope.
A microscope slide for histological or cytological use with a non-inverted optical microscope, preferably a bright field microscope, comprising a substantially flat elongated body, the body comprising a first surface and a second surface spaced from the first surface, the body comprising an aperture through the body, wherein the second surface is substantially flat and an optically transparent coverslip is mounted on the second surface to cover the aperture.
In some embodiments the microscope slide consists essentially of a substantially flat elongated body that has a first surface and a second surface spaced from the first surface, the body defining an aperture through the body, where the second surface is substantially flat and an optically transparent coverslip is mounted on the second surface to cover the aperture.
In other embodiments the microscope slide has a substantially flat elongated body which has a first sample receiving surface spaced from a second surface. In this case, the sample-receiving surface is attenuated or defines a single well and the second surface is a substantially flat optically transparent viewing surface. The attenuated surface or single well has an optically transparent flat base that has a sample of tissue for histological or cytological examination in contact with the flat base to allow the sample to be viewed through the viewing surface and the flat base so that light passing through the optically transparent flat base, sample of tissue, and layer of sealant has a reduced degree of chromatic dispersion as compared to an identically prepared control having light passing through an optically transparent flat base of at least 1 mm, sample of tissue, and layer of sealant.
In some embodiments, the sealant is optically transparent. In some embodiments, the sealant is an opaque sealant. Typically, the sealant is selectively or substantially fully light absorbing. For example, the sealant may be an opaque dark sealant. As a further example, the sealant may be an opaque black sealant. Typically, the opaque black sealant comprises a black component, such as a black dye. In embodiments where a black sealant is used, typically the microscope slide is for histological or cytological use with an epifluorescence microscope. In some embodiments, the sealant may be selected to absorb light at one or more specific wavelengths, for example to reduce background fluorescence from glass used in the slide due to using an epifluorescence microscope.
Another embodiment is directed to a non-inverted optical microscope having a stage and above the stage an objective lens. The stage supports a microscope slide having a substantially flat elongated body with a first sample receiving surface spaced from a second surface and where the second surface faces the objective lens while the sample receiving surface faces the stage and upon which a sample of tissue is placed for histological examination.
Yet other embodiments are directed to a method of preparing a tissue sample for histological or cytological examination which provides a microscope slide having a substantially flat elongated body with a first sample receiving surface that is attenuated or defines a single well and that is spaced from a second surface. Here, a tissue sample is placed in contact with the sample receiving surface of the microscope slide, the sample receiving surface is inverted and the inverted sample receiving surface is placed on a non-inverted optical microscope having a stage and objective lens so that the sample faces the stage.
Still another embodiment is directed to a kit containing a microscope slide having a substantially flat elongated body which has a first sample receiving surface spaced from a second surface. The sample-receiving surface is attenuated or defines a single well and the second surface has a substantially flat optically transparent viewing surface. The sample receiving surface's attenuated surface or single well has an optically transparent flat base.
Still another embodiment is directed to a microscope slide for histological or cytological use with a non-inverted optical microscope, said microscope slide comprising an elongated transparent body having a first surface spaced form a second surface, said first surface defining a single well in said body having a flat optically transparent base with a specimen-receiving surface adjacent to said well, said second surface being free of any wells, a specimen positioned and held in place on said specimen-receiving surface within said well, and an opaque sealant within said well to seal said specimen to said specimen-receiving surface. In some embodiments, the opaque sealant is an opaque black sealant.
The invention will now be described by way of invention only with reference to the following figures:
The inventor has realised that removing the need to view the tissue sample through the coverslip mountant would improve the resolution of the image obtained. It would also remove the need to wait long periods of time whilst the coverslip mountant solvent evaporates and the mountant polymer sets.
Accordingly one part of the invention therefore provides a non-inverted optical microscope comprising a stage and above the stage an objective lens, wherein the microscope slide comprises an optically transparent coverslip or base, having a surface facing the objective lens and a surface facing the stage, the surface facing the stage having a contact therewith a sample of tissue or cytological specimen for histological examination. That is, the sample for histological/cytological examination is viewed through the coverslip or base only and directly, rather than via a mounting material plus the coverslip or base.
Typically the tissue sample is a tissue section (i.e. “slice”) cut on a microtome, for example, a fresh tissue section or indeed, a tissue slice mounted in, for example, paraffin wax, or a cytological smear, such as a smear from a cervical examination, or a cytospin cytological sample. Cystospin samples are typically taken from, for example, sputum or by a fine needle aspirate, and cells spun down by a centrifuge onto the attenuated/thinned coverslip or base. The tissue section is typically between 2 and 7 μm thick. The tissue or cytological sample may or may not be in vitro tissue cultured cells. The tissue section or cytological sample is typically biological, e.g. animal or plant tissue and may be a medical or veterinarian biopsy or resection tissue sample.
Typically no coverslip mounting fluid is used in any aspects of the invention, although the same solvent and chemical polymers that are used for coverslip mounting may be used as a sealant.
The coverslip or base is typically 0.05-0.25 mm thick, more typically 0.1-0.2 mm thick.
Non-inverted optical microscopes such as bright field microscopes are generally known in the art.
They typically contain one or more lenses producing an enlarged image of a sample placed in the focal plane. Typically the sample is placed on a stage. The stage or indeed a body containing the lenses may be moved by the user in order to focus the image of the sample. The sample is typically illuminated from below by light focused through the stage and therefore through the sample towards the objective lenses of the microscope, through the use of, for example, an electrical light source above or below the sample that is placed on the stage.
Attempts have been made previously to try and overcome some of the problems associated with focusing on samples:
GB 1,235,587 describes a microscope slide and dark field microscope arrangement. The slides have a substantially flat elongated body having a lower surface and an upper surface spaced from the lower surface. A recess is provided in the lower surface for a transparent cover plate which provides a support for a specimen to be examined. The system attaches a sample to the underside of the coverslip and uses an immersion fluid between the sub stage lens on the microscope and the sample. The immersion fluid is substantially the same refractive index as the lens and is held in place by a passage in the stage assembly. This means that there is a risk of cross contamination between different samples used on the microscope and also risks the presence of bubbles between the sub stage lens assembly and the sample resulting in irregular lighting of the sample.
The inventor realised that viewing a sample directly through a coverslip or base, instead of via a mountant, improves the ability to study the sample.
A first aspect of the invention provides a microscope slide for histological or cytological use with a non-inverted optical microscope, comprising a substantially flat, elongated body, said body comprising a first surface and a second surface spaced from the first surface, the body defining an aperture through the body, wherein the second surface is substantially flat and an optically transparent coverslip is mounted on said second surface to cover said aperture. That is, the coverslip extends around the edges of the aperture to substantially seal the aperture. The coverslip may be presealed onto the slide, or alternatively, may have a specimen placed onto the coverslip and then the coverslip attached onto the second surface of the body with the sample within said aperture. The coverslip may be bonded to the body of the microscope slide by, for example, heat treatment or, for example, using an adhesive such as an epoxy resin adhesive that would be resistant to the various solvents that the slide might be exposed to during its pre-staining, staining and post-staining treatment. The first surface is typically substantially flat.
By having a substantially flat body, it removes the need to have a recess machined into the microscope slide for the coverslip to slot into. In one version of GB 1,235,587, the coverslip needs to be machined and placed within a recess in order to allow the microscope slide to sit properly on the stage of the microscope. By having the microscope slide of the invention being capable of being turned over, this allows the first surface of the body to sit on the stage of the microscope as a conventional microscope slide would.
The specimen is typically providing contact with the surface of the coverslip. The specimen is typically a sample of tissue for histological or cytological examination as defined above.
An alternative construction for use in the invention would be to use a microscope slide comprising a body having a first sample receiving surface spaced from a second surface, the sample receiving surface being attenuated or defining a single well, the attenuated surface or single well having a flat optically transparent base and a second substantially flat viewing surface. That is, for example, the microscope slide can simply have a well or attenuated surface machined or moulded into the slide. The sample is therefore placed within the well and the microscope slide is inverted so that the sample is viewed through the viewing surface and the base of the slide directly to the sample placed on the base. Accordingly, typically the attenuated surface or single well has a sample of tissue for histological or cytological examination in contact with the base. The viewing surface of the slide may be substantially flat across the surface of the slide, or alternatively may itself comprise a depression to allow the objective lens of the microscope to sit within the depression to allow the tissue sample to be viewed through the base.
The viewing surface closest to the objective lens, may be the first surface.
Typically the sample of tissue is as defined above.
A further aspect of the invention provides a microscope slide, the microscope slide having a first surface spaced from a second surface, the first surface being attenuated or defining a single well having a flat optically transparent base and a second surface, the second surface having mounted in contact therewith a histological or cytological sample, wherein the tissue or cytological sample is maintained in contact with the second surface by a coverslip mountant layer between the histological or cytological sample and a coverslip.
In this embodiment, the image is viewed through the well.
One of the advantages associated with the slides of the invention is that there is a reduction in chromatic dispersion compared to conventional microscope slides. In conventional microscope slides, light passes through the typically 1 mm thick glass base before interacting with the sample and subsequently travelling into the microscope. Light passing through this thick base layer of glass is chromatically dispersed and this reduces the quality of the image. In the claimed invention, a very thin layer (typically less than 100 μm) of sealant replaces this 1 mm glass layer, reducing the distance light has to travel in the medium before reaching the sample thereby reducing the degree of chromatic dispersion.
Typically the microscope slides are sized to allow them to be used with conventional prior art non-inverted optical microscopes such as bright field microscopes and epifluorescence microscopes, and/or slide scanners. Typically the microscope slides are elongated and substantially flat. Typical microscope slides are 75×25 mm and typically 1 mm to 1.2 mm thick.
The body of the microscope slide surrounding the optically transparent attenuated surface or aperture or well may be made of an optically transparent material, an optically partially transparent material or an optically non-transparent material. Accordingly the slide may be made of, for example, glass, plastics or metal. The material may be a thermoset resin. Optically transparent thermoset resins are generally known in the art.
The body of the microscope slide may be, for example, magnetic or ferromagnetic. This allows slides to be stored attached to magnetic or ferromagnetic surfaces. Use of a magnetic slide would enable the slide to attach to the microscope stage (usually made of steel) eliminating the need for the usual sprung slide holder/clip on the microscope stage.
There is no particular restriction on the type of metal from which the body of the slide is made. The metal may be an alloy or pure metal and is typically selected from steel, brass, aluminium or combinations thereof, most typically, the metal is aluminium.
Manufacturing the slide body from such materials allows the slides to be stamped out of sheet material thereby improving the ease of mass manufacture. Further, making the slide body from metal allows details to be permanently/indelibly imprinted/marked onto the slide. A portion of the microscope slide body can be imprinted with information, such as sample numbers, using various printing techniques, avoiding the use of sticky labels or glass pens which can fall off, be smudged or washed away during use or storage. Typical printing/marking processes include dot matrix stamping or laser engraving or etching.
Typically the optically transparent attenuated surface or base or coverslip is made of glass or plastic. Optically transparent thermoset resins are generally known in the art. Typically the coverslip or base is as defined above.
A single attenuated surface or aperture or well is typically provided per slide.
The attenuated area or single well or aperture will be of a sufficient size to permit the application of a histological or cytological sample and may be offered in a range of sizes to accommodate different sample sizes (e.g. biopsy sample versus larger resection specimen sample).
The attenuated surface, aperture or well may be square, rectangular, round, oval or indeed substantially any shape in the plane of the microscope slide.
The specimen or tissue sample may be protected and/or kept in place by the use of a sealant, such as a rapid setting sealant such as a thermoset epoxy resin or a thermoplastic nail varnish-type polymer. In some embodiments, the sealant comprises one or more thermoplastic acrylic polymers. In some embodiments, the sealant comprises one or more polymers or copolymers selected from the group consisting of poly(ethyl methacrylate), poly(methyl methacrylate), poly(ethyl acrylate), a copolymer of methyl methacrylate with ethyl acrylate, a copolymer of methyl methacrylate with ethyl methacrylate, and combinations thereof.
As the tissue sample is not necessarily illuminated through the sealant (i.e. the tissue sample may be illuminated through a transparent coverslip/base/attenuated area, or by using reflected light) it is possible to use non-optically transparent sealants. Where the tissue sample is illuminated through the sealant (as in a standard optical microscope), the sealant will, of necessity, be translucent. The sealant may be, for example, fluorescent or chemiluminescent. The use of a UV lamp, for example, may allow the sealant to fluoresce and illuminate the tissue sample.
In some embodiments, the sealant is applied to the slide as a liquid. In some embodiments, the sealant is applied to the slide by spraying as an aerosol. This allows a thin film sealant to be applied evenly onto a sample and therefore improves transmission and reduces dispersion of light through the slide so as to yield a better quality image than conventional slides. In some embodiments, the thickness of the film will be less than 100 μm and in some embodiments the film has a thickness in the range 10 μm to 100 μm. In some embodiments, the film will have a thickness in the range 20 μm to 80 μm. In some embodiments, the film has a thickness of about 50 μm.
There is no particular restriction on the type of sealant used in the invention however it is preferred that sealant has low viscosity, a similar refractive index to the materials used in the window of the slide (typically glass), and dries quickly. In some embodiments, the sealant is optically transparent. In some embodiments, the sealant comprises distyrene as this has a refractive index very similar to glass. In some embodiments, the sealant is non-optically transparent, such as an opaque sealant (e.g., an opaque black sealant). The sealant typically includes a solvent to facilitate application of the sealant to the slide which can subsequently evaporate leaving behind the other sealant components as a thin film. Other components can also be added to improve the film forming or optical properties of the sealant such as dewetting agents to minimise the formation of a meniscus which can act as a lens, distorting images.
Examples of resins include compositions comprising distyrene, a plasticiser and xylene.
The microscope slides of the invention may comprise an indicium on the coverslip (where the coverslip is subsequently applied to the body of the slide by the user and therefore requires identification of the attached tissue specimen or cytological preparation separate from the body of the slide) or the second surface of the body or alternatively or additionally an indicium on the first surface of the body or the coverslip. Indiciums can be used to uniquely or non-uniquely identify the coverslip or the microscope slide to which the coverslip is attached. The indicium may, for example, be a barcode, or other machine-readable code. An example of an indicium on a coverslip is shown in US 2007/0092408. Alternatively, it may be a roughened area to allow the slide or coverslip to be easily written on.
As the microscope slide may at different stages be rotated so that different sides of the slides face the user, it is important to ensure that the tissue or cytological sample attached to the slide is immediately identifiable. Accordingly, typically an indicium is provided on both sides of the microscope slide when in use. Accordingly, the indicium may be provided on one or both sides of the coverslip and/or on the one or both surfaces of the body of the microscope slide. This allows the microscope slide to be inverted and the origin/identity of the tissue sample, to be readily determined from both sides.
The microscope slide may be used in combination with a non-inverted optical microscope such as bright field as discussed above.
The coverslip or base may be coated with, for example, lysine to allow the sample to better adhere.
Methods of preparing a tissue sample for histological or cytological examination are also provided comprising providing an optically transparent attenuated surface or coverslip or base, placing the tissue sample in contact with the surface of the attenuated surface or coverslip or base, inverting the whole microscope slide and placing it on the stage of a non-inverted, bright field optical microscope, the non-inverted, bright field optical microscope comprising a stage, an objective lens, wherein the sample faces the stage and wherein either:
(a) the coverslip is mounted on a second surface of a body, comprising a first surface and a second surface spaced from the first surface, the body defining an aperture through the body, wherein the second surface is substantially flat and the coverslip is mounted on the second surface with the sample within the aperture; or
(b) the base forms part of a sample receiving surface of a microscope slide for histological use comprising a body, said body comprising a viewing surface and a sample receiving surface defining a single well, the single well comprising an optically transparent base.
The tissue may be as defined above.
Tissue samples may be mounted in wax, such as paraffin wax and a section cut using a microtome. Typically this section is floated on a water bath from where the section is placed on the coverslip or base. The tissue section then may be heated, for example, to approximately 65° C. to adhere the section to the surface of the base or coverslip. The wax may then be removed using solvents such as xylene and alcohols, prior to staining, as generally known in the art.
Alternatively, tissue samples may be cryomounted using techniques generally known in the art, sliced into sections and placed on the coverslip or base prior to staining. The inventors have found that the claimed invention is particularly useful in electrodeposition. Without being bound by theory, it is thought that the thin window or well base in the claimed invention is capable of storing a greater static charge than conventional glass slides. This is particularly useful when looking at frozen sections as when the slide of the invention is brought near to a sample, the sample jumps into the well or onto the charged window (depending upon which surface of the slide the sample is placed). Restricting the window to a specific portion of the slide body also means that the window acts as a target to consistently guide samples to a particular portion of the slide. This is sometimes a problem when preparing frozen sections with large charged glass slides.
A still further aspect of the invention provides a microscope slide kit comprising a body, the body comprising a first surface and second surface spacer on the first surface the body defining an aperture through the body, and the second surface is substantially flat; and an optically transparent attenuated surface or coverslip or thin base.
A kit additionally comprising one or more of a sample sealant, a stain and/or a coverslip adhesive may also be provided. Typically, the adhesive is a chemically and/or heat resistant material. Typically, the adhesive is a resin that is UV-curable.
The use of the coverslip mountant, with the provision to seal the aperture with a traditional coverslip mountant solution (which, being exposed to the atmosphere without the overlying coverslip as per its traditional use, will dry more quickly) or a fast drying sealant such as an epoxy resin, or indeed a fast-drying clear nail varnish, has the advantage of avoiding sticky slides, which is problematic if they stick to paperwork, slide trays etc. There is no need to wait, for example, 48 hours for traditionally covered slides to dry before they can be filed. They can be filed shortly after viewing by which time the fast-drying, curing epoxy resin or fast drying solvent based polymer sealant has already been applied.
Furthermore, because the sealant is exposed to the air rather than being sandwiched between the glass base and the glass coverslip (as in a traditional microscope slide), there is no formation of bubbles. This also improves the quality of sample images.
Provision of wells allows reagents to be easily used by placing the reagents into the well to contain them. Alternatively, the slide or racked slides can be immersed in pots of histological or cytological staining reagents to stain the tissue section/sample or cytological preparation.
The slides are typically compatible with slide racks, staining machines and existing microscopes and digital microscope scanners. This avoids the need for new laboratory equipment to be provided.
The resultant higher resolution image of the tissue or cytological sample achieved will be of benefit in the interpretation of the sample. The resultant higher resolution image of the tissue or cytological sample will improve the quality of the scanned digital image resulting from the use of a microscope slide digital scanner.
The provision of the sealant and attenuated area or well design avoids the need for an automated coverslipping machine.
The aperture sealant may additionally be adapted, for example, by provision of a reflective, chemiluminescent or chem-uv-luminescent material to improve or modify the background light for the sample.
The microscope slides may also be used with a digital microscope slide scanner instead of an optical microscope. Such scanners produce a digitised image of the microscope slide.
In use, the tissue section is viewed through the microscope lens (20), through the coverslip (18) and the coverslip mountant (16). Typically this produces the blurred/out of focus results shown on the left hand side of
The use of stain is optional.
The tissue section may be held in place and protected, for example, with using a sealant, such as an epoxy resin or indeed a film-forming polymer such as nitrocellulose dissolved in butyl acetate or ethyl acetate (i.e. “nail varnish”). In
The sealant stage can dry rapidly either by the use of a fast-curing adhesive such as an epoxy resin or the use of a solvent based sealant open to the atmosphere allowing the solvent (e.g. xylene or toluene) to evaporate quickly thus permitting the material to dry quickly. The dried microscope slide comprising the tissue section will then be inverted prior to placing on a microscope.
A still further embodiment of the invention is shown in
A conventional slide of the prior art is shown in
The microscope slide configuration according to the present invention enables optimum microscope image quality of the specimen. Spherical aberration is a well-recognised phenomenon that occurs when light comes into focus in different planes rather than in the same plane, decreasing microscope image resolution and intensity. Spherical aberration most commonly occurs when the specimen sits further away from the coverslip surface through which the light path travels, as the light will refract through the sealant. This refraction will cause the light to come into focus in different planes resulting in a deviation/distortion from the optimal spherical shape of the focussed light as it comes in and out of focus. Spherical aberration is therefore significantly reduced when using a microscope slide of the invention where the specimen is affixed directly to the coverslip through which the specimen is viewed, compared to using the standard microscope slide set up where sealant is required to secure the coverslip over the specimen.
It is also important to note that the standard microscope slide surface is not as flat as that of a coverslip surface because glass of microscope slides is made by the float glass method whereas coverslips are made by the draw glass method. This can lead to the specimen affixed to the standard microscope slide being in multiple different planes, and therefore, more out of focus than a sample affixed to a coverslip glass. A sample affixed to the flatter surface of a coverslip glass (as is the case for the microscope slide of the invention) has more of the specimen present in one plane, leading to more of the specimen being in focus on the specimen image. An example of an image demonstrating this effect is found in
The unique configuration of the slide according to the invention provided an opportunity for further improvement with respect to the sealant. The sealant may be optically transparent or non-optically transparent.
In some embodiments, the sealant comprises one or more thermoplastic acrylic polymers. In some embodiment, the sealant comprises about 15% (w/w) to about 30% (w/w) of one or more thermoplastic acrylic polymers. In some embodiment, the sealant comprises about 20% (w/w) to about 25% (w/w) of one or more thermoplastic acrylic polymers. In some embodiments, the sealant comprises one or more polymers or copolymers selected from the group consisting of poly(ethyl methacrylate), poly(methyl methacrylate), poly(ethyl acrylate), a copolymer of methyl methacrylate with ethyl acrylate, a copolymer of methyl methacrylate with ethyl methacrylate, and combinations thereof. In preferred embodiments, the sealant comprises a copolymer of ethyl methacrylate and methyl acrylate.
In preferred embodiments, the resultant sealant film (i.e., the dried layer of sealant on the coverslip) has a refractive index very similar to the refractive index of the specimen and the coverslip glass. In some embodiments, the resultant sealant film has a refractive index of between about 1.47 and about 1.54. In some embodiments, the resultant sealant film has a refractive index of between about 1.48 and about 1.52. In some embodiments, the resultant sealant film has a refractive index of about 1.49. In some embodiments, the coverslip has a refractive index of between about 1.50 and about 1.54. In some embodiments, the coverslip has a refractive index of between about 1.51 and about 1.53. In some embodiments, the coverslip has a refractive index of about 1.52. In preferred embodiments, the sealant will not substantially yellow or deteriorate in optical clarity over time.
Typically, the sealant comprises one or more solvents. This facilitates application of the sealant to the slide as the solvent(s) subsequently evaporates leaving behind the other sealant components as a thin film. In some embodiments, the sealant comprises about 60% (w/w) to about 90% (w/w) or one or more solvents. In some embodiments, the sealant comprises about 65% (w/w) to about 85% (w/w) or one or more solvents. In some embodiments, the sealant comprises about 70% (w/w) to about 80% (w/w) or one or more solvents. In preferred embodiments, the sealant comprises acetone and xylene which provides an optimal viscosity with an optimal drying time (i.e., slow enough to prevent air entrapment and fast enough to ensure that hardening occurs before the specimen is microscopically assessed). If the viscosity of the sealant is too high, the sealant will not spread evenly, and a resultant uneven film can affect the optical properties of the image. In some embodiments, the sealant comprises about 60% (w/w) to about 90% (w/w) acetone and about 1% (w/w) to about 8% (w/w) xylene. In some embodiments, the sealant comprises about 65% (w/w) to about 80% (w/w) acetone and about 3% (w/w) to about 6% (w/w) xylene.
Typically, the sealant mixture is applied to the specimen using a pipette. Typically, about 0.5 to about 1 mL of sealant is applied to the aperture of the slide. In preferred embodiments, about 0.6 to about 0.8 mL of sealant is applied to the aperture of the slide. In particularly preferred embodiments, about 0.7 mL is applied to the aperture of the slide. This provides a resultant hardened film of a thickness between about 0.13 and about 0.17 mm
In some embodiments, the sealant is optically transparent. The optically transparent sealant may be used for viewing the slide by standard transilluminating light microscopy (e.g., bright-field microscopy). The optically transparent sealant may also be used for viewing the slide using other microscopy techniques, such as epifluorescence microscopy.
An example of an optically transparent sealant formulation is provided in the table below:
In alternative embodiments, the sealant is opaque (i.e., non-optically transparent). For example, the sealant may be an opaque black sealant (i.e., light absorbing). This sealant is particularly suitable for use with the microscope slide of the invention when specimens are to be viewed using epifluorescence light microscopy. In this type of microscopy, the excitation ultraviolet light path is not transmitted through the sample, but rather travels through the objective lens, through the coverslip glass to the specimen, where fluorophores are excited to emit light in the visible wavelength. Some of this specimen-emitted fluorescent light travels back into the objective lens to the eye or camera detector.
It will be clear to the person skilled in the art that an optically clear sealant could also be used when viewing the sample with epifluorescence light microscopy; however, an opaque (e.g., black) sealant is preferred as this eliminates background autofluorescence (e.g., from deep within the specimen) as well as stray light and improves the image contrast. It will also be clear to the person skilled in the art that an opaque sealant cannot be used with a standard microscope slide and coverslip arrangement. This is because the black sealant would necessarily need to be applied on top of the specimen (as in
The black sealant typically has equivalent physical behaviour to that of an optically transparent sealant but is non-optically transparent to light in the visible spectrum because of the inclusion of a black component. Preferably, the black component disperses and/or dissolves into the polymer-solvent mixture to create an opaque black film when fully hardened. In some embodiments, the black component is a black pigment or dye. In preferred embodiments, the black component is a molecular dye, such as Sudan Black B or carbon black. This provides the advantage that the dye disperses and dissolves within the solvent (e.g., a xylene/acetone solvent mix) and provides a uniform and highly opaque black film. Typically, the black component is Sudan Black B.
In some embodiments, the sealant comprises about 0.1 to about 10% (w/w) of the black component (e.g., black dye). In some embodiments, the sealant comprises about 0.2 to about 9% (w/w) of the black component (e.g., black dye). In some embodiments, the sealant comprises about 0.3 to about 8% (w/w) of the black component (e.g., black dye). In some embodiments, the sealant comprises about 0.4 to about 7% (w/w) of the black component (e.g., black dye). In some embodiments, the sealant comprises about 0.5 to about 5% (w/w) of the black component (e.g., black dye). In some embodiments, the sealant comprises about 0.5 to about 4% (w/w) of the black component (e.g., black dye). In some embodiments, the sealant comprises about 0.5 to about 3% (w/w) of the black component (e.g., black dye). In some embodiments, the sealant comprises about 0.5 to about 2% (w/w) of the black component (e.g., black dye). In some embodiments, the sealant comprises about 0.5 to about 1% (w/w) of the black component (e.g., black dye).
An example of a black sealant formulation is provided in the table below:
In some embodiments, the specimen is stained with one or more immunofluorescent markers (i.e., labels or tags). For specimens stained with an immunofluorescence marker that will be assessed using an epifluorescence microscope, application of the black sealant behind the specimen fixed to a slide of the present invention has multiple benefits. These benefits include prevention of light ingress to the specimen which in turn reduces signal fading and photobleaching but most importantly eliminates background autofluorescence which maximises the image contrast, resulting in improved optical clarity. An improved image obtained using the slide of the invention and black sealant is provided in
In some embodiments, the sealant (optically transparent or opaque) can be processed into a solid form for benefits in storing and transporting the material. Supply of the sealant as a solid eliminates storage and transportation of controlled chemicals such as acetone and xylene. For example, a black sealant can be originally prepared at the manufacturing facility using a solvent which can then be removed through solvent removal routes such as heat and/or use of a vacuum. The resultant solid can be cooled immediately and then undergo further processing to obtain a desired particle size. The resultant solid may be supplied in any suitable form, such as a block, pellets, granules, or powder. In the end-user's laboratory, the solid form can then be redispersed with the preferred ratio solvents, returning the formulation to a liquid form for pipetting into the slide's specimen-fixed window.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1403822.8 | Mar 2014 | GB | national |
This nonprovisional application is a Continuation-In-Part of co-pending application No. 16/549,363 filed on Aug. 23, 2019, which is a Continuation application of application No. 15/123,370, now abandoned, which was filed on Sep. 2, 2016, which was the National Phase Under 35 USC § 371 of PCT International Application No. PCT/GB2015/050617 filed on Mar. 4, 2015, which claims priority under 35 U.S.C. § 119 on Patent Application No. 1403822.8 filed in the United Kingdom on Mar. 4, 2014, the entire contents of each of which are hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15123370 | Sep 2016 | US |
| Child | 16549363 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16549363 | Aug 2019 | US |
| Child | 18309680 | US |
