WO2014071959A1

WO2014071959A1 - Cover slip materials for microscopy

Info

Publication number: WO2014071959A1
Application number: PCT/EP2012/004630
Authority: WO
Inventors: Régis Robert BARBIEUX; Martine Renée BOUVET; Carine Gabrielle LEFÈVRE; Jean-Christophe Fernand Minor
Original assignee: Morgan Adhesives Company
Priority date: 2012-11-07
Filing date: 2012-11-07
Publication date: 2014-05-15

Abstract

The present invention relates to cover film materials useful for preparing specimens for microscopy and methods of making and using the same.

Description

COVER SLIP MATERIALS FOR MICROSCOPY

TECHNICAL FIELD OF THE INVENTION

The present invention relates to cover film materials useful for preparing specimens for microscopy and methods of making and using the same. BACKGROUND OF THE INVENTION

In biological disciplines such as histology and pathology it is common to examine specimens such as tissue samples using light microscopy. Preparing a specimen for microscopic examination includes mounting the specimen onto a glass slide, optionally fixing and/or staining the specimen, and further covering the specimen with a cover slip. Traditionally, cover slips are made from very thin, transparent glass to ensure adequate light transmission. However, thin glass cover slips have many disadvantages including their fragility and expense, as well as their impracticability to be used in automated slide preparation equipment.

Cover slips of polymeric materials that are less brittle than glass and are suitable for semi-automated or automated slide preparation are known. For example, to permit automation of the task of applying cover slips to specimen slides, the polymeric film is supplied in the form of a roll that can be fed automatically into a machine capable of applying the film as a cover slip to specimen slides. To further aid specimen

examination and preservation, a sealing agent or primer resin can be coated onto the cover slip film and can be activated with a small amount of solvent when applied to the specimen slide. For example, W099/53357 and U.S. Patent Nos. 4,455,188;

4,188,246; 3,939,019; 4,146,414; 4,203,797; 4,853,262; 4,171 ,866, 4,607,921;

5,580,414, 5,406,421 , 5,590,414 and 3,498,860 disclose slides, cover slips and/or apparatus for applying cover slips to slides. Although these products allow for semi- automation or automation of specimen preparation, the cover slip materials have several disadvantages including cracking of the primer and separation of the primer from the substrate or backing film, blocking of the film on a large roll, as well as inadequate adhesion of the primer. These issues can lead to automation interruption, inadequate sample preservation, poor optical quality of the final preparation, as well as l specimen loss. Commercially available polymeric coverslips are available, for example, from Sakura Finetek under the name Tissue-Tek™ Coverslipping Film, and KP Tape from Klinipath Corporation of Germany.

Therefore, improved cover slip materials suitable for semi-automated or automated specimen slide preparation are highly desired.

SUMMARY OF THE INVENTION

The present invention provides an improved cover slip material suitable for use in semi- automated or automated specimen slide preparation equipment.

In one aspect, the present invention provides a light-transmissible cover slip material comprising a light-transmissible polymeric substrate having a first planar surface and a second planar surface, and a multi-layer bonding layer disposed on the second planar surface wherein the bonding layer further comprises a first layer of at least one pressure sensitive adhesive and a second layer of at least one methacrylate polymer.

Especially, according this aspect of the present invention, there is provided a light- transmissible cover slip material as defined in claim 1 ; further, particularly advantageous embodiments of the inventive cover slip material are the subject-matter of the respective dependent claims.

In a second aspect, the present invention provides a method of making a light- transmissible cover slip material comprising coating a pressure sensitive adhesive layer onto one planar surface of a light-transmissible polymeric substrate, drying the coated pressure sensitive adhesive layer, coating a silicon web casting sheet with a

methacrylate polymer to form a methacrylate polymer layer, laminating the methacrylate polymer layer onto the pressure sensitive adhesive layer, and removing the silicon web casting sheet. It is self-explanatory that any explanations, details, embodiments etc. discribed in the following for one aspect of the present invention also refers to the other aspect accordingly even without specific mention in order to avoid any kind of repetitions. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts a cross-sectional view of an embodiment of the material of the present invention.

Figure 2 depicts a perspective view of a specimen slide incorporating an embodiment of the material of the present invention.

Figure 3 depicts a cross-sectional view of the specimen slide and an embodiment of the material of the present invention of Figure 2.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a light-transmissible cover slip material useful for preparing specimens for microscopic examination.

Referring to Figure 1 , shown is a cross-section view of one embodiment of the material of the present invention. The light transmissible cover slip material 14 comprises a light-transmissible polymeric substrate 13 having a first planar surface 16 and a second planar surface 17. A layer of pressure sensitive adhesive 12 is disposed onto the second planar surface 17 of the polymeric substrate 13. A layer of methacrylate polymer 11 is laminated to the layer of pressure sensitive adhesive 12. Bonding layer 15a comprises pressure sensitive layer 12 and methacrylate polymer layer 11.

Referring to Figure 2, glass microscope slide 20 is shown with specimen 25 mounted thereon. Specimen 25 is in view through one embodiment of the light-transmissible cover slip material 14 of the present invention.

Figure 3 is a cross-section side view of the same assembly as shown in Figure 2.

Cover slip material 14 as in Figure 1 is applied and adhered to glass slide 20 over specimen 25 with the aid of an activating solvent such as xylene. Following exposure to and evaporation of the xylene, a bonding layer 15a of Figure 1 is transformed into a bonding layer 15b of Figure 3. Transmission of light through the cover slip material must be in an amount sufficient for the eye or camera that is viewing the specimen. Therefore, maximum transmittance of light passing through the cover slip material is desired. In one embodiment, the transmittance of the cover slip material is at least 85% according to ASTM D1003-95. In another embodiment, the transmittance is at least 90%. In a further embodiment, the transmittance is at least 93%.

As used herein, the term "polymer" means a generally high molecular weight chemical compound made up of smaller molecules or monomers. The monomers that make up the polymer can be the same, as in a homopolymer, or different, as in a copolymer. As used herein the term "polymer" refers to homopolymers as well as copolymers.

In accordance with the present invention, the light-transmissible polymeric substrate can be any polymeric substrate with adequate flexibility and transmissibility for use in automated or semi-automated specimen preparation. In one embodiment, the substrate has a thickness from about 50 microns to about 150 microns. In another embodiment, the substrate has a thickness of about 110 microns to about 120 microns. In a specific embodiment, the substrate has a thickness of about 115 microns. Many polymeric substrates are commercially available and are suitable for use in the present invention. For example, the substrate can be selected from cellulose triacetate, cellulose diacetate, polyethylene terephthalate, styrene-acrylonitrile and polymethyl methacrylate films. In one embodiment, the substrate is an acetate film. In a particular embodiment, the substrate is cellulose diacetate.

The pressure sensitive adhesives (PSA) that are coated on the second planar surface of the substrate can be any pressure sensitive adhesive having at least the following characteristics: (a) has adequate clarity or transmittance of at least 85%; (b) is capable of creating lasting adhesion to the substrate or backing layer of the cover slip and the glass slide while preserving a specimen in combination with the methacrylate layer of the present invention for at least ten years; (c) is at least partially soluble in an activating solvent such as xylene; and (d) does not yellow or lose substantial clarity following evaporation of the activating solvent through a ten year period. The composition of the pressure sensitive adhesive can vary based on the particular application. In certain embodiments, cross-linked PSA compositions are preferred. In one embodiment, the PSA comprises an acrylic resin and a cross-linking agent. Examples of such PSA formulations include those available commercially from Henkel sold under the brand name of DuroTak®. As used herein, the phrase "glass transition temperature" or "Tg" means the

temperature at which an amorphous material reversibly transitions from a hard and relatively brittle state into a molten or rubber-like state as defined by, for example, ISO 11357-2: Plastics - Differential scanning calorimetry (DSC) - Part 2: Determination of glass transition temperature (1999). In addition to the properties mentioned above, the PSA of the present invention can have a glass to liquid transition temperature of about - 60°C to about +5°C. In one embodiment, the Tg of the PSA is from about -40°C to about 0°C. In another embodiment, the PSA has a Tg of about -20°C to about -5°C.

The pressure sensitive adhesive can be coated onto the light-transmissible polymeric substrate using conventional techniques known in the art, such as solvent coating by methods such as reverse roll, knife-over-roll, gravure, wire wound rod, floating knife or air knife, hot-melt coating such as by slot orifice coaters, roll coaters or extrusion coaters, at approximate coating weights. Alternatively, a UV activated initiator can be included and the monomer mixture may be partially cured by UV exposure into a viscous liquid. The viscous liquid can then be coated by any conventional technique and additional UV exposure can be provided to obtain the final desired properties. In one embodiment of the present invention, the pressure sensitive adhesive is coated onto the second planar surface of the polymeric substrate at an amount of about 5.0 to about 50.0 g/m². In another embodiment, the PSA is coated onto the substrate at an amount of about 10.0 to about 30.0 g/m². In another embodiment, the PSA is coated onto the substrate in an amount of about 15.0 to about 25.0 g/m². In yet another embodiment, the PSA is coated onto the substrate at an amount of about 20.0 g/m².

The methacrylate layer in accordance with the present invention can comprise one or more polymers. Examples of polymers useful in the present invention include isobutyl methacrylate, methacrylate, methyl methacrylate, n-butyl methacrylate and

combinations thereof. Commercially available polymers and resins include DEGALAN PQ611 , DEGALAN PM381 N, ELVACITE 2013, ELVACITE 2028, and ELVACITE 2550. (DEGALAN brands are available from Evonik of Germany, and ELVACITE brands are available from Lucite International, Inc.) In one embodiment, the methacrylate layer is a blend of isobutyl methacrylate and methyl methacrylate polymers. The methacrylate layer can be prepared as a thin film that can be laminated to the pressure sensitive adhesive layer of the material. For example, the methacrylate layer can be a blend of methyl methacrylate polymer and methacrylate polymer that is formed into a layer on a siliconized casting web and heated to aid in drying. The layer, once dried, can be laminated to the pressure sensitive adhesive layer and the casting sheet can then be removed. Processes such as these for preparing thin films for lamination are known in the art. In one embodiment, the methacrylate blend is applied to the casting web at about 2.0 to about 30.0 g/m². In another embodiment, the methacrylate blend is applied to the casting web at about 5.0 to about 20.0 g/m². In a further embodiment, the methacrylate blend is applied to the casting web at about 5.0 to about 10.0 g/m².

In one embodiment, the cover slip material of the present invention is wound into a roll. In another embodiment, the material is wound into a roll with the methacrylate layer on the outside of the roll. In a preferred embodiment, the roll of material can withstand storage conditions at 23°C +/- 2°C and 50% relative humidity +/- 5% without blocking. The cover slip material can be produced in a variety of widths and lengths as

determined by the available equipment for production and processing as well as the size of the automated or semi-automated equipment for preparing specimen slides. In one embodiment, material is produced in a width of about 1.5 meters and then is cut into a narrower width for storage and distribution. The length of the roll can be manufactured in virtually any length, including lengths of over 1000 meters, and cut to shorter lengths if desired for shipping and storage, as well as for use in the specimen producing equipment. In one embodiment, the rolled material is about 24 mm in width and about 70-75 meters in length. The material of the present invention can be used in a process for preparing a specimen for microscopic evaluation. The process for preparing such specimen comprises placing a specimen on a glass slide and applying a sufficient amount of activating solvent to the slide, covering the specimen with a cover slip material of the present invention while exposing the bonding layer of the cover slip material to the activating solvent to at least partially solubilize the bonding layer of the cover slip material, and further adhering the cover slip material to the specimen and the slide upon evaporation of the activating solvent. In one embodiment of the present invention, the activating solvent is xylene. Use of activating solvents such as xylene to adhere cover slip materials coated with soluble polymers to glass slides is known in the art. Employing adhesives to adhere cover slip materials to glass slides is also known and has been described, for example, in W099/53357. However, these materials and their associated uses have undesirable outcomes such as cracking of the polymeric layer after bonding, blocking of the material following storage on a roll, separation of the covers slip material from the slide or cover slip substrate after extended storage, and poor adhesion of the cover slip material resulting in damage or loss of the specimen. The inventors have found that employing a multi-layer bonding layer comprising a pressure sensitive adhesive coated with a layer of xylene-soluble polymer can provide superior adhesion properties with little or no cracking of the polymer layer following evaporation of the activating solvent, and further allows for storage of the cover slip material in a roll without blocking. Not being bound by any particular theory, it is believed that upon exposure of the multi-layer bonding layer to the xylene activating solvent, the

methacrylate layer of the multi-layer bonding layer at least partially dissolves and can expose and possibly even cause a sufficient amount of the pressure sensitive adhesive layer to dissolve in the xylene, transforming the multi-layer bonding layer of the cover slip material into a transformed bonding layer that provides enhanced adhesion to the slide following evaporation of the activating solvent. The exact structure of the newly transformed bonding layer following xylene exposure and evaporation is not precisely understood. However, the transformed bonding layer surprisingly not only provides superior adhesion of the bonding layer to the substrate and the glass slide, but the transformed bonding layer (i.e. the pressure sensitive adhesive layer in combination with the methacrylate layer following xylene exposure and evaporation) does not experience significant hazing or cracking such as that typically seen with existing methacrylate/xylene solvent coverslip systems.

The above description and the following examples illustrate certain embodiments of the present invention and are not to be interpreted as limiting. Selection of particular embodiments, combinations thereof, modifications, and adaptations of the various embodiments, conditions and parameters normally encountered in the art will be apparent to those skilled in the art and are deemed to be within the spirit and scope of the present invention.

EXAMPLES

Materials and Methods

180° Peel Adhesion: Peel adhesion is measured according to Finat FTM-1 (peel adhesion 180° at 300 mm per minute) at the end of each week for eight weeks. Slides can be prepared by placing the cover slip material on a glass slide in the presence of about 120μΙ of xylene and further evaporating off the xylene. The results are shown in Table 1.

Folding Resistance: Folding resistance is subjectively measured by bending the cover slip material sample to 180° and further examining the material for fissures or separation of the bonding layer from the substrate. Folding resistance is subjectively measured as follows: +/- = the methacrylate layer fissures but is maintained in place by the pressure sensitive adhesive layer; NOT OK = the methacrylate layer cracks and separates from the polymeric substrate; OK = the methacrylate layer remains unchanged without any fissures or separation. Results are shown in Table 2. Tappi T569 Separation Test: Internal bond strength (Scott type) of the cover slip material is measured using the Tappi T569 method. The separation being measured is the separation of the bonding layer from the substrate or backing layer in accordance with the Tappi T569 standardized test. Results are shown in Table 2.

Haze: Haze of the cover slip material is measured prior to application of the cover slip to the slide and measurements are in accordance with ASTM D-1746. Results are shown in Table 2.

Clarity: Clarity of the cover slip material is measured prior to application of the cover slip to the slide and measurements are in accordance with ASTM D-1476. Results are shown in Table 2. Light Transmittance: Light transmittance is measured after application of the cover slip to the slide and measurements are in accordance with ASTM D1476. Results are shown in Table 2. EXAMPLE A. The cover slip material is prepared as follows: a cellulose diacetate substrate with a thickness of approximately 115 microns is coated with a cross-linkable acrylic pressure sensitive adhesive (PSA) of the following formulation: 86.93 wt% cross- linkable acrylic resin containing 35.5 wt% solids in a solvent of 37.0 % ethyl acetate, 22.0 % isopropanol, and 41 % heptane, 1.08 weight % cross-linking agent,

(commercially available DuroTak 180-122A/R40 cross-linker) and 11.99 wt% butyl acetate solvent. The PSA formulation is coated onto one side of the substrate at a thickness of approximately 20g/m² and dried at 90°C to evaporate the solvents. The methacrylate layer is prepared by slowly mixing until substantially dissolved 28.6 wt% isobutyl methacrylate and 14.3 wt% methyl methacrylate in 57.1 wt% toluene. The mixture is coated onto a pre-siliconized casting sheet web at 7.0g/m² with a Mayer rod, and application is transverse to the machine direction of the casting web. The

methacrylate layer is dried at 80°C to facilitate polymerization. The methacrylate layer is laminated to the PSA layer surface of the PSA-coated acetate film, taking care not to entrap any air between the two layers. The casting web is then removed.

EXAMPLE B. The cover slip material is prepared as in Example A, with the exception that the cross-linkable acrylic pressure sensitive adhesive (PSA) is of the following formulation: 87.12 wt% cross-linkable acrylic resin containing 35.5 wt% solids in a solvent of 37.0 % ethyl acetate, 22.0 % isopropanol, and 41 % heptane, 0.87 weight % cross-linking agent, and 12.02 wt% butyl acetate solvent.

EXAMPLE C. The cover slip material is prepared as in Example A, with the exception that the cross-linkable acrylic pressure sensitive adhesive (PSA) is of the following formulation: 87.31 wt% cross-linkable acrylic resin containing 35.5 wt% solids in a solvent of 37.0 % ethyl acetate, 22.0 % isopropanol, and 41 % heptane, 0.65 weight % cross-linking agent, and 12.04 wt% butyl acetate solvent.

EXAMPLE D. The cover slip material is prepared as in Example A, with the exception that the cross-linkable acrylic pressure sensitive adhesive (PSA) is of the following formulation: 87.5 wt% cross-linkable acrylic resin containing 35.5 wt% solids in a solvent of 37.0 % ethyl acetate, 22.0 % isopropanol, and 41 % heptane, 0.43 weight % cross- linking agent, and 12.07 wt% butyl acetate solvent. EXAMPLE E. The cover slip material is prepared as in Example A, with the exception that the cross-linkable acrylic pressure sensitive adhesive (PSA) is of the following formulation: 87.78 wt% cross-linkable acrylic resin containing 35.5 wt% solids in a solvent of 37.0 % ethyl acetate, 22.0 % isopropanol, and 41 % heptane, 0.11 weight % cross-linking agent, and 12.11 wt% butyl acetate solvent.

EXAMPLE F. The cover slip material is prepared as in Example A, with the exception that the cross-linkable acrylic pressure sensitive adhesive (PSA) is of the following formulation: 100.0 wt% cross-linkable acrylic resin containing 35.5 wt% solids in a solvent of 37.0 % ethyl acetate, 22.0 % isopropanol, and 41 % heptane, without cross- linking agent or solvent.

EXAMPLE G. The cover slip material is prepared as in Example D, with the exception that the methacrylate layer is prepared by slowly mixing until substantially dissolved 28.6 wt% isobutyl methacrylate and 14.3 wt% methyl methacrylate/n-butyl methacrylate polymer in 57.1% toluene? The mixture is coated onto a pre-siliconized casting sheet web at 7.0g/m² with a Mayer rod, and application is transverse to the machine direction of the casting web. The methacrylate layer is heated at 80°C to facilitate drying.

EXAMPLE H. The cover slip material is prepared as in Example A, with the exception that the pressure sensitive adhesive is obtained and prepared as commercially available DuroTak pressure sensitive adhesive. COMPARATIVE 1. Klinipath coverslip tape that can be obtained from Klinipath of Germany.

COMPARATIVE 2. TissueTek® coverslipping film that can be obtained from Sakura.

Table 1 FINAT FTM-1 Peel adhesion (180S) N/inch

1 week 2 week 3 week 4 week 5 week 6 week 7 week

Example A 9.03 16.67 14.19 13.64 13.62 9.63 8.65

Example B 10.12 18.66 19.08 13.82 12.31 18.05 21.15

Example C 10.08 16.62 17.44 18.55 17.6 16.59 18.9

Example D 7.71 12.92 14.11 21.6 19.3 18.89 24.14

Example E 8.11 17.38 18.94 20.98 21.96 22.3 14.55

Example F 2.33 15.75 20.09 20.28

Example G 11.64 16.29 17.83 19.85 20 21.56 21.09

Example H 4.5 12.69 12.39 7.81

Comparative

0.75 1.59 2.71 1.08 0.97 4.28 2.65 Example 1

Comparative

10.78 14.04 13.19 18.96 12.7 19.16 18.2 Example 2

Table 2

Claims

Claims:

1) A light-transmissible cover slip material comprising: a. a light-transmissible polymeric substrate having a first planar surface and a second planar surface; and b. a multi-layered bonding layer disposed on said second planar surface, wherein the bonding layer further comprises: i. a first layer of at least one pressure sensitive adhesive; and ii. a second layer of at least one methacrylate polymer.

2) The material of claim 1 , wherein the polymeric substrate is an acetate film. 3) The material of claim 2, wherein the acetate film is cellulose diacetate.

4) The material of claim 2 or 3, wherein the thickness of the acetate film is about 50 microns to about 150 microns.

5) The material of claim 4, wherein the thickness of the acetate film is about 110 microns to about 120 microns. 6) The material of claim 5, wherein the thickness of the acetate film is about 115 microns.

7) The material of any of claims 1 to 6, wherein the methacrylate polymer is

selected from isobutyl methacrylate, methyl methacrylate, and a combination thereof. 8) The material of any of claims 1 to 7, wherein the pressure sensitive adhesive is a cross-linkable acrylate pressure sensitive adhesive.

9) The material of claim 8, wherein the pressure sensitive adhesive further

comprises a. one or more cross-linkable acrylate polymers; b. a cross-linking agent; and c. a solvent.

10) The material of any of claims 1 to 9, wherein the pressure sensitive adhesive has a Tg of about -20°C to about -5°C. 11) The material of claim 10, wherein the pressure sensitive adhesive has a Tg of about -7°C.

12) The material of any of claims 1 to 11 , wherein the pressure sensitive adhesive first layer of the bonding layer is disposed directly onto the second planar surface of the substrate. 13) The material of claim 12, wherein the pressure sensitive adhesive is disposed onto the substrate in an amount of about 15 to about 25 g/m².

14) The material of any of claims 1 to 13, wherein the second layer of the bonding layer is disposed onto the first layer of the bonding layer.

15) The material of any of claims 1 to 14, wherein the methacrylate second layer of the bonding layer is in an amount of about 5.0 to 10.0 g/m².

16) The material of any of claims 1 to 15, wherein the second layer of the bonding layer is partially or completely soluble in an activating solvent.

17) The material of any of claims 1 to 16, wherein the first layer of the bonding layer is partially or completely soluble in an activating solvent. 18) The material of claim 16 or 17, wherein the activating solvent is xylene.

19) The material of any of claims 1 to 18, wherein the material has a transmittance of at least 85%.

20) The material of claim 19, wherein the material has a transmittance of at least 90%. 21) The material of claim 20, wherein the material has a transmittance of at least 93%.

22) The material of any of claims 1 to 21 , wherein the material has a 180° peel

adhesion of at least 2.0 N/inch after one week. 23) The material of claim 22, wherein the material has a 180° peel adhesion of about 6.0 to about 12.0 N/inch after one week.

24) The material of any of claims 1 to 23, wherein the material has a 180° peel

adhesion after eight weeks that is greater than or equal to the 180° peel adhesion of the material after one week. 25) The material of any of claims 1 to 24, wherein the material has a 180° peel

adhesion of at least 4.0 N/inch after eight weeks.

26) The material of claim 25, wherein the material has a 180° peel adhesion of about 7.0 to about 25.0 after eight weeks.

27) The material of any of claims 1 to 26, wherein the material has a separation

value greater than 1050 J/m² in a Tappi T569 separation test.

28) The material of any of claims 1 to 27, wherein the haze of the material is less than 27 in accordance with ASTM D-1746.

29) The material of claim 28, wherein the haze of the material is less than 20 in

accordance with ASTM D-1746. 30) The material of any of claims 1 to 29, wherein the clarity is at least 30 in

accordance with ASTM D-1746.

31) The material of claim 30, wherein the clarity is at least 39 in accordance with ASTM D-1746.

32) The material of any of claims 1 to 31 , wherein the material is in the form of a roll. The material of claim 32, wherein the material is wound in a roll wherein the second layer of the bonding layer is on the outside of the roll.

A method of making a cover slip material according to any of claims 1 to 33, comprising: a. coating the pressure sensitive adhesive layer onto the second planar layer of the substrate; b. drying the substrate and the pressure sensitive adhesive; c. coating a silicon web casting sheet with a methacrylate polymer to form a methacrylate polymer layer; d. laminating the methacrylate polymer layer to the pressure sensitive

adhesive layer; and e. removing the silicon web casting sheet.

The method of claim 34, wherein the method is characterized by one of more of the features comprised by any of claims 1 to 33.