EP1721141A1

EP1721141A1 - Means for covering microscopic preparations

Info

Publication number: EP1721141A1
Application number: EP05707120A
Authority: EP
Inventors: Guenter Hauke; Daniela Grabis; Andreas Beirau; Heinz Allmann
Original assignee: Merck Patent GmbH
Current assignee: Merck Patent GmbH
Priority date: 2004-03-04
Filing date: 2005-02-01
Publication date: 2006-11-15
Also published as: JP2007526463A; WO2005085799A1; US20070166197A1

Abstract

The invention relates to covering glasses which are coated with a covering medium and which are used to cover preparations and to mechanical methods for the production thereof. The inventive covering glasses make it significantly easier to cover preparations by virtue of the fact that it is not necessary to handle the glutinous covering medium and the fact that the preparations can be firmly covered in just a few minutes and can be microscoped using immersion lenses. The layer thicknesses and layer thickness tolerances according to the invention result in a stable and lasting covering.

Description

Means for mounting microscopic specimens

The invention relates to coverslips coated with mounting medium, processes for their production and their use in the manual and automatic mounting of microscopic coverslips

Preparations.

The microscopic examination of specimens in transmitted light technology is based on the refraction of light on very thin objects. For this purpose, the specimens are first drawn onto a slide, treated with reagents, such as staining reagents or dewatering agents, and then firmly surrounded with a glass-like resin. This is done by embedding or infiltrating resins in the samples. These resins, also called mounting media, penetrate the thin section of the test material and ideally combine without

Phase break with the slide under the material and the cover slip over the material. In this way, a preparation is obtained which is embedded in a glass phase and which is well suited for examination under a transmitted light microscope and which is also preserved.

Glass is generally used as the preferred material for slides and cover glasses. However, it is also possible, for example, to use a plastic film or plastic cover glasses instead of a cover glass to cover the preparations. In contrast to glass, however, these materials have the disadvantage that they are not completely impermeable to solvents. The covered specimens age faster and the cover film can peel off.

Preparations can be mounted in hydrophobic or hydrophilic mounting media. To cover in hydrophobic media, the preparation must first be dewatered after it has been drawn onto the slide and stained. For this purpose, it is finally introduced into xylene or a xylene substitute via an ascending alcohol series. The slide is then provided with the hydrophobic mounting medium and that

Cover glass so that there are no air bubbles in the specimen. After the mounting medium has hardened, the preparation is sealed airtight in a glass-like environment and can be examined with a transmitted light microscope.

The sample does not have to be dewatered for mounting in hydrophilic mounting media, but can be provided with the mounting medium immediately. After covering with the coverslip and hardening of the mounting medium, the preparation is sealed airtight in a glass-like environment, as when using a hydrophobic mounting medium, and can be examined under a transmitted light microscope.

Covering the preparations is a very complex and time-consuming step. If the mounting medium is applied manually to the slide, care must be taken that a constant amount is always applied, otherwise the medium will emerge and stick to the side, or the covering may not be complete. Even when using automatic coverslipping devices, malfunctions often occur, since escaping mounting medium sticks to the machine. If the covered specimens are stacked too quickly after covering or are stored tightly, they can also stick to one another or to their surroundings.

The mounting medium also requires drying times of at least 30 minutes before curing, and even more than 45 minutes for hydrophilic media. In this way, long waiting times have to be accepted before the embedded preparation can be examined under the microscope. This applies in particular to investigations with immersion objective. The large amount of time required is also a major disadvantage, particularly in the case of rapid cuts, which are removed, for example, during an operation and must be examined immediately.

US 3,498,860 and EP 1242800 disclose the use of cover glasses which have previously been coated with mounting medium or adhesive. The advantage of this method is that the cover slip can be coated with the mounting medium beforehand, dried and stored until use. There is no need to work with liquid mounting media during mounting. The drying times are also greatly reduced.

Nevertheless, the method disclosed in US 3,498,860 and EP 1242800 was not used in practice, since the coated cover glasses showed only moderate adhesive properties and often detached again after a short time. The object of the present invention was therefore to provide cover glasses coated with mounting medium which do not have the disadvantages of the prior art.

It has been found that cover glasses coated with mounting medium can be produced with very good adhesive properties and a long service life if the layer of mounting medium is between 0.05 and 0.8 mm and the layer thickness tolerance is not more than ± 0.1 mm, preferably no more than + 0.05 mm. Coatings with these properties cannot be reliably produced manually. However, it has been found that mechanical processes enable the cover glasses to be produced with the desired coating.

The invention therefore relates to a means for covering preparations, which essentially consists of a cover glass and a layer of covering medium applied thereon, characterized in that the layer thickness of the covering medium is between 0.05 and 0.8 mm and the layer thickness tolerance is not more than ± 0.1 mm.

In a preferred embodiment, the layer thickness tolerance ^{5 is} not more than ± 0.05 mm.

In a preferred embodiment, the layer thickness of the mounting medium is approximately 0.2 mm.

^" 1 ° In a preferred embodiment, the cover slip is made of glass.

In a preferred embodiment, the solids content of the mounting medium in the dried state is 150-300 mg over an area of 24 × 50 mm. 15 The present invention also relates to a process for the production of coverslips coated with mounting medium, characterized in that the mounting medium is machined by means of one or more metering needles, by means of printing processes, coating by means of a liquid curtain, application by a slot nozzle, distribution by means of a doctor blade or by means of Spin coating with a layer thickness between 0.05 and 0.8 mm and a layer thickness tolerance of no more than + 0.1 mm is applied to the cover slip. ⁵ In a preferred embodiment, the mounting medium is applied using one or more dispensing needles.

In another preferred embodiment, the mounting medium is applied by means of screen printing. 0 The invention also relates to the use of a cover glass according to the invention coated with mounting medium for the manual or automatic mounting of preparations.

According to the invention, the covering of specimens means the provision of a specimen slide on which there is an opened, optionally colored specimen wetted with an intermediate as well as a cover glass coated according to the invention and the subsequent covering of the specimen slide with the cover glass. When covering with hydrophobic mounting media, the last solvent in the drainage series or staining, i.e. typically xylene or xylene substitutes, typically called water when covered with hydrophilic mounting media. The preparations can be stored in these solvents for a longer period until they are covered.

The cover slip according to the invention greatly simplifies the mounting of preparations. While three components, i.e. the slide with the mounted specimen, the liquid mounting medium and the cover slip must be brought together, the method according to the invention is based on two easy-to-use components, i.e. reduced the slide with the drawn-up preparation moistened with intermediate and the coated cover glass.

According to the invention, the term coverslip includes all coverslips known and suitable for the purpose, e.g. Cover glasses made of glass or plastic or foils. Cover glasses made of glass are preferred according to the invention. Depending on their size, these are preferably provided on one side with a defined amount of the liquid mounting medium.

In a preferred embodiment of the present invention, one side becomes one side of a cover slip complete with a layer a mounting medium. However, it is also possible that the layer does not completely cover the cover slip, but rather leaves the edge areas free. It is particularly important that the cover glass is covered with the covering medium layer according to the invention at the points at which the specimen to be microscoped is located. The layer of mounting medium must be so much larger than the surface of the specimen that it extends beyond the specimen on all sides and thus creates a firm bond with the slide on which the specimen is located.

All mounting media typically used for mounting according to the prior art can be used as mounting medium for coating the cover glasses. The coating can be carried out with both hydrophobic and hydrophilic mounting media. These are, for example, mounting media based on organic polymers, such as Canada balm, acrylate resins, e.g. Polymethacrylate, polystyrene, polyvinyl pyrrolidone, polyvinyl alcohol or glycerol gelatin. Typically, mounting media of this type contain solvents, such as water, xylene or xylene substitutes, such as toluene, or other aromatic or aliphatic hydrocarbons, and further additives which are known to the person skilled in the art. Further examples of mounting media can be found in "Romeis - Microscopic Technology", Urban & Schwarzenberg, 17th edition, 1989, pages 296-297.

It was found that the solids content of the mounting media for

Coating of the cover glasses should be approximately 40% or more. However, this value is only to be understood as a guideline, but not as an absolute value. This value can change depending on the respective solid component, the surface tension and the viscosity of the mounting medium. The expert in the field of coverslipping is able to do this after a few coating and coverslipping tests assess which solids content or which volume of mounting medium per mm ² coverslip makes sense for the respective mounting medium.

The viscosity of the commercially available mounting media can be increased by evaporating part of the solvent before use, so that the solids content increases. Mounting media with a viscosity between 2000 and 4000 mPas have proven to be particularly suitable. The use of mounting media with a viscosity of around 3000 mPas is particularly advantageous. It is difficult to process higher-viscosity media with the layer thickness and layer thickness tolerance to be achieved.

The solids content of the mounting medium should be between 150 and 300 mg in an area of 24 x 50 mm when dry. Approx. 200 mg are preferably applied to an area of 24 x 50 mm. If the solids content is higher, there is a risk that the layer thickness is so great that the solvent does not penetrate the layer or does not penetrate the layer quickly enough, and then the slide and cover glass are not bonded. If too little is applied, the polymer mass is insufficient and the functionality is not guaranteed.

It was found that a critical size of the coated cover glasses for a stable and permanent covering is in particular the layer thickness of the mounting medium and the layer thickness tolerance.

If the layer thickness fluctuates over a tolerance of ± 0.1 mm, the cover glass often detaches from the specimen at some points after a short time. As a result, the preparation ages prematurely and can no longer be examined microscopically. Optimal storage stability and durability were found for coatings with a thickness between 0.05 and 0.8 mm, preferably between 0.1 and 0.4 mm, and a layer thickness tolerance of no more than ± 0.1 mm, preferably no more as ± 0.05 mm, particularly preferably not more than ± 0.02 mm. Such a coating can only be applied with difficulty and not reliably by hand. However, it was found that various mechanical processes are well suited for the even application of the mounting medium to the cover glasses:

Application using a dispensing needle (s)

Application using one or more dispensing needles is particularly preferred. With this method, a full-surface as well as partial surface coating of the materials to be coated is possible.

The mounting medium is placed in a container and applied to the cover slip, for example with the aid of a metering pump, compressed air, liquid pressure or by means of a piston, via one or more metering needles. The coating takes place by the metering needle and / or the cover slip or the template with the cover slip moving in the x and y directions. The corresponding homogeneous surface and layer thickness can be controlled via the speed of the movement of the needle or the cover slip in the x / y direction and / or via the throughput from the needle. The advantage of this process is that the mounting medium can be precisely adjusted in terms of viscosity beforehand and remains in a closed system until it is discharged (no change in viscosity during the coating process). The diameter of the dispensing needle is typically between 0.25 and 7.5 mm, preferably about 0.8 mm. It should be noted that the distance between the needle and cover glass must be within very tight tolerances - according to the tolerances of the layer thickness. The distance between the dispensing needle and the cover slip should correspond to the desired layer thickness of the mounting medium. The coating can be optimized for each mounting medium by the speed of the dispensing needle, the spacing of the individual coating lines, the volume flow of the mounting medium and the viscosity of the mounting medium. If necessary, the needle and / or the mounting medium can be tempered.

printing process

Printing techniques are also suitable for coating the cover slips, foils and plates. The most important are: Pad printing Offset printing Gravure printing Screen printing InkJet

The screen printing method has proven to be a particularly suitable method.

Here it is possible to work with highly viscous mounting media and thus apply defined layer thicknesses. The screen can also be designed so that only the desired surfaces are coated. In order to ensure a large number of pieces and good utilization of the large-format machines, the cover glasses must be placed in a carrier. This can take many tiles. It must be designed in such a way that cavities are provided in which the cover slips fit exactly, so that there is a flat surface for printing. Since high forces occur during printing, the cover glasses are preferably fixed with vacuum support.

The mounting medium is then placed on the screen and the printing process is then started.

Typically, a filler knife spreads the medium over the entire sieve. Then the squeegee moves over it. This expresses the filled

Sieve, which is a distance of a few millimeters to the cover glasses has on this. This will print the mounting medium on the coverslips. The layer thickness depends exclusively on the sieve used. The open mesh size and the thread diameter of the sieve must be adapted to the respective mounting medium, in particular to its viscosity. As a rule, sieves with mesh sizes between 300 and 1500 μm, preferably between 500 and 1000 μm, and thread diameters between 100 and 500 μm are suitable. For example, with a mounting medium with a viscosity of 2900 mPas with a sieve

'' with a width of 688 μm and a thread diameter of 140 μm achieves a moist layer thickness of 200 μm. By drying, the layer thickness becomes a little smaller. Frequently, especially with mounting media with a relatively low viscosity, it is advantageous not only to carry out a screen printing coating, but rather two or more, e.g. with 5 a carousel screen printing process. After printing, the carrier with the cover glasses is removed and a new process can be prepared.

Coating with a liquid curtain 0 The coverslips are pushed under a liquid curtain. The layer thickness is determined by the speed of the glasses and the volume flow of the mounting medium.5 For this process, the covering medium has to be diluted very much to ensure a uniform flow. It can therefore be advantageous to carry out several coating passes. 0 The advantages of this method are the particularly uniform surface that can be achieved in this way. Apply through a slot nozzle

In this process, the mounting medium is pressed with pressure through a slot nozzle that has the exact same width as the cover glasses. By moving the nozzle, the entire surface is scanned and thus that

Medium brought onto the glass.

The nozzle can be designed so that the medium is brought directly from the nozzle onto the glass, or in such a way that the medium is spread by a device after it has hit the glass surface.

Punctual application of the mounting medium and distribution with a slide (doctor blade)

The mounting medium is applied to the cover glass at one point, analogous to the manual process. Then it is spread with a slide, a so-called squeegee. This can be a spiral doctor blade, which is guided directly on the glass surface, or a plate, which is guided at the distance that the desired layer thickness corresponds to.

With this method, an exact layer thickness is easy to achieve. However, some mounting medium is always pushed away to the side. Therefore a carrier is required for coating, in which the cover glasses are inserted. This too must be cleaned after each coating run. In addition, the plates must always be separated from the carrier to dry, since they can no longer be removed after the medium has hardened. Spin coating

In this process, the cover slip to be coated rotates. The mounting medium is applied in drops and is distributed through the

5 centrifugal force. Since some medium always leaves the surface of the glass, the spin coating machine must be constantly cleaned here, otherwise contamination and sticking of the glasses cannot be ruled out.

10 As a rule, each of the methods mentioned is followed by a drying step after application of the mounting medium. If the coated coverslips are to be used immediately, a drying time of 1 to 5 minutes is sufficient, preferably at a slightly elevated temperature in the

, ₅ drying cabinet. Temperatures that are too high, ie temperatures above 60-80 ° C, are not suitable for many mounting media because the polymer is attacked and cracks can occur in the coating. If the coverslips are to be stored and, if necessary, stacked directly on top of one another, then longer drying times are possible up to 24

_{? n} hours beneficial. For example, 2 hours at 60 ° C in a drying cabinet have proven to be very suitable.

For the covering, the coated cover glass with the coated side is placed on a slide which is still wet with intermediate

₂₅ known technology launched. Due to the coating with the mounting medium, the cover glass and slide combine within a few seconds and harden. Typically, the preparation is completely cured after only 3 minutes, usually even after less than one minute, and can then be used, for example, using immersion

₃₀ objective, microscopic. Because of the simple handling, the coated cover slip according to the invention is suitable for both manual and automatic mounting of specimens. Since no liquid mounting medium has to be used for mounting, there is no risk of sticking. The specimens fixed on the slides are preferably removed immediately after staining and optionally after dewatering from the last solution (intermediate), ie for example water or xylene or xylene substitutes, and covered in a still moist state. If the preparations are already almost dry, they should be moistened again with a little intermediate.

Another parameter to be considered is the type and thickness of the preparations. The cover slip according to the invention is suitable for covering preparations, for example from histology and cytology, as are typically also covered according to the prior art.

The cover glasses according to the invention are therefore particularly suitable for covering specimens with a thickness of up to 15 μm. Depending on the thickness of the cover glass coating and the type of mounting medium, thicker preparations can also be covered.

In addition to manual covering, the coated coverslips and the method according to the invention can also be used for automatic covering with automatic coverslipping machines. Coverslipping machines which, according to the prior art, wet the slides with a defined amount of mounting medium and then put on the coverslip are commercially available. For the coverslipping method according to the invention, both coverslippers according to the prior art, the function of which is slightly modified, and coverslippers that are designed for the method according to the invention can be used. Conventional automatic coverslipping machines have a metering pump to apply liquid mounting medium to the slide. This is not necessary in the method according to the invention. Accordingly, this can Step are omitted when using automatic coverslippers for the method according to the invention. Another possibility is to use this metering device to place some intermediate medium (water, xylene, etc.) on the slide instead of the mounting medium. Since the specimen slides with the specimens for the method according to the invention should still be moist, specimens that have already dried slightly after staining or dewatering could be moistened again with a small amount of intermediate. The preparation is then covered with the cover slip according to the invention.

In another embodiment of an automatic coverslipper, which is particularly suitable for the method according to the invention, the specimen slides with the specimens are stored in a container filled with solvent (intermediate). The slides are only transported out of the solvent container for covering and are directly covered with the coated cover glass. The supply of mounting medium or other amounts of solvent is not necessary here.

By using the cover slip according to the invention, the coverslipping machines are treated much more gently than with conventional methods, since no sticky coverslipping medium can get into the machines. Just like with manual mounting, the specimens can be microscoped after a short time.

The cover glasses according to the invention and the method according to the invention for producing these cover glasses now offer the possibility of covering preparations quickly and easily. Manual handling of sticky mounting media is eliminated. A permanent covering is generated by the layer thickness and layer thickness tolerance of the mounting medium according to the invention. The covered specimens can be stored for a long period of time without the cover glass coming off. Even without further explanations, it is assumed that a person skilled in the art can use the above description in the broadest scope. The preferred embodiments and examples are therefore only to be regarded as descriptive, in no way as in any way limiting in any way.

The complete disclosure of all of the applications, patents and publications mentioned above and below, in particular the corresponding application EP 04005103.9, filed on March 4, 2004, is incorporated by reference into this application.

Examples

1. Coating using screen printing

A sieve with an open mesh size of 688 μm and a thread diameter of 140 μm is stretched on a frame of 700 * 500 mm and mounted on a flatbed screen printing machine.

6 * 7 rows of coverslips 25 * 50 mm are positioned on a support on a support using vacuum.

The mounting medium with a viscosity of 2900 mPas is placed on the screen, the screen is lowered over the cover glasses and then the mounting medium is pressed through the screen onto the cover glasses using a doctor blade.

Immediately afterwards, the sieve is raised again. Then the

Cover glasses removed for drying.

2. Coating using a dispensing needle

The cover slips (25x50 mm) are placed on a device using a device

Vacuum table positioned. The diameter of the dispensing needle is 0.8 mm. Entellan ^® with a viscosity of 2900mPas is used as mounting medium.

A syringe with a dispensing needle is filled and closed with a plunger.

The distance between the dispensing needle and cover glass should correspond to the layer thickness to be applied (here: 0.2 mm)

The dispensing needle is positioned over a cover glass using a dispensing robot. The syringe plunger is then pressurized with approx. 2 bar and the needle is moved at a speed of 60-100mm / min.

Individual "mounting medium beads" are applied as a Z-shape. With this procedure, the cover glasses are coated individually.

Claims

Expectations

1. Means for mounting preparations, which essentially consists of a coverslip and a layer of mounting medium applied thereon

5, characterized in that the layer thickness of the mounting medium is between 0.05 and 0.8 mm and the layer thickness tolerance is not more than ± 0.1 mm.

2. Means according to claim 1, characterized in that the 10 layer thickness tolerance is not more than ± 0.05 mm.

3. Composition according to claim 1 or 2, characterized in that the layer thickness of the mounting medium is approximately 0.2 mm.

^ ⁵ 4. Agent according to one or more of claims 1 to 4, characterized in that the cover glass is made of glass.

5. Composition according to one or more of claims 1 to 4, characterized in that the solids content of the mounting medium in the dried state on an area of 24 x 50 mm is 150-300 mg.

6. Process for the production of coverslips coated with mounting medium, characterized in that the mounting medium is machined by means of one or more dispensing needles, by means of a printing process, coating by a liquid curtain, application by a slot nozzle, distribution by means of a doctor blade or by means of spin coating with a layer thickness between 0 , 05 and 0.8 mm and a layer thickness tolerance of no more than ± 0.1 mm is applied to the cover slip ⁰ .

7. The method according to claim 6, characterized in that the mounting medium is applied by means of one or more dispensing needles.

8. The method according to claim 6, characterized in that the mounting medium is applied by means of screen printing.

9. Use of an agent for covering preparations according to one or more of claims 1 to 5 for manual or automatic covering of preparations.