DE20214835U1 - New carrier for a biological sample, to be cut by laser micro-dissection, comprises film which is absorbent to laser light forming the base of a Petri dish - Google Patents

Abstract

A carrier for a biological sample, to be cut by laser micro-dissection, is on a freely clamped film which is absorbent to laser light, and fitted to a frame holder. The holder is generally the wall of a Petri dish, with a base which is wholly or partially missing to be replaced by the film, in a thickness of 1.35 Microm or 2.5 Microm, welded to the Petri dish wall or bonded by an adhesive.

Description

B 3204 EN
24.09.2002

Carrier device for a biological, laser microdissection

cuttable preparation

The invention relates to a carrier device for a biological preparation that can be cut by laser microdissection, which is arranged on a freely stretched laser light-absorbing film that is applied to a frame-shaped holder.

In the field of biology and medicine, microdissection is a process in which a small piece is cut out of a generally flat specimen (e.g. cells or a tissue section) using a fine, focused laser beam. The cut-out piece is then available for further biological or medical (e.g. histological) examinations. The type of specimen preparation depends, among other things, on the laser microdissection process to be used to process the specimen.

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From DE 201 00 866.1 a carrier device for a preparation, in particular for a biological preparation, is known, wherein the preparation is intended for cutting out a preparation area by means of a focused laser beam. The carrier device has a laser light-absorbing and thus laser-cuttable film for holding the preparation, wherein the film is applied to a frame-shaped holder so that it is freely stretched, i.e. not supported or carried by further carrier means below the film. This carrier device for a preparation is specially designed to be used in a method for laser microdissection, in which the cutting, focused laser beam is directed from above onto the

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preparation is aligned and the cut-out preparation area falls down after the cutting process.

Such a method has already been described in the article "Cell surgery by laser micro-dissection: a preparative method", G. Isenberg, W. Bielser, W. Meier-Rüge, E. Remy, Journal of Microscopy, Vol. 107, May 1976, pages 19-24. A focused laser beam from a pulsed UV laser is directed from above onto a specimen, preferably a biological one, and a specimen region of interest is surrounded by the focused laser beam along a closed cutting line. This completely separates the specimen region of interest from its surroundings and causes it to fall down into a collection device.

DE 100 43 506 describes a device and a newer method for laser microdissection. A focused laser beam is directed from above onto a specimen, preferably a biological specimen. In a first step, a specimen region of interest is surrounded by the focused laser beam along an open cutting line that largely does not enclose the specimen region of interest, with a stable bridge remaining between the beginning and end of the cutting line, via which the specimen region of interest is connected to the surrounding sample. In a second step, the bridge is cut through with a single focused laser pulse directed at the bridge, with the cutting width previously adjusted to the width of the bridge, i.e. increased. With the last cutting laser pulse, the specimen region of interest is completely separated from its surroundings and falls down. The method has the advantage over the previously mentioned method that it prevents the almost cut-out specimen region from folding away or twisting towards the end of the cutting process.

The previous application areas of laser microdissection consisted of the selection of cells from histological sections, e.g. in molecular pathology, cell biology and neuroscience. However, users increasingly want to be able to select cells from

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a culture or collection of living cells. For cell culture, the above-mentioned carrier devices must therefore be placed in a Petri dish. The films of the carrier devices are then wetted or coated with culture medium and the desired cells are seeded onto them. After the cells have grown, the carrier devices are removed from the Petri dish and placed in a device for laser microdissection, as described in DE 100 43 506.

The disadvantage of the carrier devices is that the cells can only be kept alive for a short time after the carrier device has been removed from the Petri dish. In addition, the film of the carrier devices can be damaged during handling. Furthermore, the device for laser microdissection can become contaminated because the carrier device also comes into contact with culture medium in the area of its frame or underside, so that cells can also colonize there. When examining pathogens, this not only presents a handling problem, but also a hygiene problem.

It is therefore an object of the present invention to provide a carrier device which allows a laser microdissection of living cell cultures in a comfortable and hygienic manner, in particular with a laser beam directed onto the preparation from above.

This object is achieved by a carrier device for a biological preparation that can be cut by means of laser microdissection, which is arranged on a freely stretched laser light-absorbing film that is applied to a frame-shaped holder, wherein the carrier device is characterized according to the invention in that the frame-shaped holder is essentially designed as the wall of a Petri dish with a completely or partially missing bottom and that instead of the missing bottom, only the laser light-absorbing film is arranged.

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The carrier device according to the invention has the advantage that the carrier device itself is used for pre-culturing the cells, which simplifies cell pre-cultivation. After cultivation, the cells are also under optimized, reliable growth conditions during laser microdissection. The handling problem is eliminated because it is no longer necessary to remove the cell cultures from the Petri dish, as was previously the case. Contamination of the device used for laser microdissection is also ruled out.

Various designs of the support device are possible. For example, the entire base of the Petri dish can be formed by the laser light-absorbing film. However, it is also conceivable to form only part of the base of the Petri dish by the laser light-absorbing film. For example, the wall of the Petri dish and an edge area of the base can be made of plastic and only a remaining opening in the base of the Petri dish is closed by the film. It is crucial that the film is laser light-absorbing, i.e. laser-cuttable, and that the film is stable enough not to sag in the spanned base opening and to support the culture medium including the cells. The layer thickness of the film must therefore be sufficiently thick.

A polyethylene naphthalate film (PEN) has therefore proven to be suitable for the laser light-absorbing film of the carrier device, which preferably has a thickness of 1.35 μm or 2.5 μm. However, other film thicknesses can also be used depending on the application.

The connection between the wall of the Petri dish and the laser light-absorbing film can be realized in different ways. For example, the laser light-absorbing film can be welded to the lower edge of the wall of the Petri dish or the edge area of the opening in the bottom of the Petri dish.

B 3204 EN 24.09.2002

An inexpensive solution is to glue the wall of the Petri dish to the laser light-absorbing film. The bonding can be done using an adhesive tape. The adhesive tape is preferably designed in the form of a template so that the adhesive tape is glued to the wall of the Petri dish on one side and to the laser light-absorbing film on the other side.

In another embodiment of the connection between the wall of the Petri dish and the laser light-absorbing film, the film can be pre-prepared on ring-shaped holding elements, i.e. applied using welding or adhesive technology. The diameter of the ring-shaped holding elements is matched to the cylindrical wall of the Petri dish. The ring-shaped holding elements have locking grooves which allow the wall of the Petri dish to snap into place, so that a liquid-tight, removable connection is created between the wall of the Petri dish and the ring-shaped holding element with the laser-cuttable film base. This embodiment has the advantage that the ring-shaped holding element with the laser-cuttable film base can be separated from the wall of the Petri dish again, so that the cell culture can either be further processed or, for example, archived or frozen in a space-saving manner.

For handling in the laboratory, it is advantageous if the laser light-absorbing film is designed to be hydrophilic, as this makes it easier to apply a cell culture medium to the laser-cuttable film. A nutrient liquid is usually used as the nutrient medium. Nutrient media for cell culture are commercially available, for example DMEM (Dulbeco's Modified Eagle's Medium) or RPMI (Rosewell Park Memorial Institute Medium) or MEM.

Depending on the size of the cut-out preparation areas, the nutrient fluid can pass through the microscopically small holes created in the laser-cuttable film during laser microdissection.

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seep through. It is therefore particularly advantageous if the nutrient medium is designed as a nutrient gel that is sufficiently solid or at least very viscous so that it "stays" around the holes created by microdissection in the laser-cuttable film.

The desired preparation area can be separated by moving the focused laser beam around the preparation area of interest along a closed cutting line. After the preparation area of interest has been completely separated from its surroundings by the laser beam, it falls down and can be collected in a collecting device.

In an alternative cutting method, a specimen area of interest is surrounded by a focused laser beam along an open cutting line that largely does not enclose the specimen area of interest. A stable bridge remains between the beginning and end of the cutting line, via which the specimen area of interest is connected to the surrounding sample. In a second step, the bridge is cut through with a single focused laser pulse directed at the bridge, the cutting width of which is adjusted to the bridge. This completely separates the specimen area of interest from its surroundings and causes it to fall down.

The carrier device according to the invention allows the use of a method for laser microdissection on living biological cell cultures, in which a focused laser beam is directed from above onto a living biological preparation. The cells are treated particularly gently because they fall into a collection vessel by gravity after being cut out. Mechanical or laser-induced transport of the cells, which carries the risk of cell damage, is thus superfluous.

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Areas of application include the selection of preferably living cells or organisms from pure cultures or mixed cultures in order to subject them to further analysis or cultivation. For example, cancer cells can be separated from a group of healthy cells, from stained cells from cultures, from microorganisms from mixed cultures (or cultures) or from parasites from cultures.

Advantageous embodiments of the invention are explained below with reference to the schematic drawing. They show:

Fig. 1. a first embodiment of a carrier device with a full-surface film base;

Fig. 2. a second embodiment of a carrier device with a non-full-surface film base;

Fig. 3. a third embodiment of a carrier device with a reversibly attachable full-surface film base;

Fig. 4. a device for laser microdissection with a carrier device with a full-surface film base;

Fig. 1 shows a first embodiment of a carrier device 1 with a full-surface film base. Fig. 1a shows the carrier device 1 in a vertical section. Fig. 1b shows the carrier device 1 from below.

The carrier device 1 has a frame-shaped holder which is designed as the wall 2 of a Petri dish, which is typically made of plastic. The Petri dish has no bottom. The missing bottom of the Petri dish is replaced by a laser-light-absorbing and thus laser-cuttable film 3 which is glued to the lower edge 4 of the wall 2. The adhesive is only applied in a thin layer and is therefore not shown. It is crucial that the adhesive connection is resistant to a nutrient solution or a viscous nutrient gel for the cell culture to be grown.

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In addition, it must be ensured that the adhesive is not cytotoxic so that the biological preparation is not damaged.

The absorption of the laser-cuttable film 3 is adapted to the wavelength of the laser intended for cutting, with a pulsed UV laser preferably being used for laser microdissection. The use of a polyethylene naphthalate film (PEN), which preferably has a thickness of 1.35 μm or 2.5 μm, has therefore proven to be useful for the laser light-absorbing film 3 of the carrier device 1. However, other film thicknesses can also be used depending on the application.

In order to enable problem-free cutting with the focused laser beam later, the film must be applied exactly flat and without ripples. Only then is it possible, once the focus of the laser beam has been adjusted, to cut at different points on the film by simply moving the laser beam and the carrier device relative to each other, so that a closed cutting line can be created.

A biological preparation 5 is applied to the laser-cuttable film 3. In the present example, it is a living biological preparation. This was obtained by wetting or coating the laser-cuttable film 3 of the carrier device 1 with culture medium. The desired cells were seeded onto it. The decisive factor here is that the thickness of the laser-cuttable film 3 is sufficient to support the preparation and the culture medium without bending. Only then is it later possible, as described above, to precisely cut the flat film 3 in a single focus setting of the laser beam. The preparation 5 was created by the growth of the cell culture. The carrier device 1 together with the preparation 5, i.e. the cell culture, can now be placed in a device for laser microdissection.

Fig. 2 shows a second embodiment of a carrier device 1 with a non-full-surface foil base. Fig. 2a shows the carrier device 1 in

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a vertical section. The carrier device 1 has a frame-shaped holder which is designed as a wall 2 of a Petri dish and also forms an outer ring-shaped part 6 of the bottom of the Petri dish.

Fig. 2b shows the support device 1 from below. In this illustration, it is clearly visible that the Petri dish does not have a closed bottom. Instead, there is only an outer ring-shaped part 6 of the bottom of the Petri dish, which encloses a free opening 7. The opening 7 is spanned and thus closed by a laser light-absorbing and thus laser-cuttable film 3, which is glued to the underside of the ring-shaped part 6 of the bottom of the Petri dish. In this way, the laser-cuttable film 3 replaces the missing bottom of the Petri dish. In the present example, the bonding was realized using a suitably shaped adhesive film 8.

Fig. 2c shows a top view of an embodiment of this suitably shaped adhesive film 8. It is pre-shaped in a template-like manner so that it completely encloses the free opening 7. A small tab 9 makes handling easier.

The adhesive film 8 can preferably be a double-sided adhesive tape, in which adhesive is applied to both sides of a solid, non-detachable carrier material, which is covered on the outside with a cover film. After peeling off the first cover film, the adhesive tape with the exposed adhesive can be placed at the desired location on the underside of the ring-shaped part 6 of the base of the Petri dish. The second cover film is then also peeled off, so that the adhesive tape with its carrier material remains on the ring-shaped part 6 of the base. The laser-cuttable film 3 can then be applied to the exposed adhesive of the adhesive tape and glued to it.

Alternatively, a solid adhesive film applied between two stable cover films can be used as the adhesive film 8. After removing the first cover film, the adhesive film can be applied to the desired location on the underside of the ring-shaped part 6 of the bottom of the Petri dish.

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placed. The second cover film is then also removed so that only the adhesive film remains on the ring-shaped part 6 of the base. The laser-cuttable film 3 can then be applied to the adhesive film and glued to it.

The decisive factor for all adhesives is that the adhesive is resistant to a nutrient solution or a viscous nutrient gel that is to be applied later for the cell culture to be grown. In addition, the adhesive bond must withstand the weight of film 3, nutrient medium and cell culture. In addition, it must be ensured that the adhesive film is not cytotoxic so that the biological preparation is not damaged.

Fig. 3 shows a further embodiment of a carrier device 1 with a reversibly attachable full-surface film base. Fig. 3a shows the carrier device 1 in a vertical section. The carrier device has a frame-shaped holder which is designed as the wall 2 of a Petri dish. The Petri dish has no base. The missing base of the Petri dish is replaced by a laser light-absorbing and thus laser-cuttable film 3.

In contrast to the embodiment in Fig. 1, the laser-cuttable film 3 is not glued directly to the lower edge of the wall 2. Instead, the carrier device additionally has an annular holding element 10, to the underside of which the laser-cuttable film 3 is glued. On its upper side, the annular holding element 10 has a circumferential locking groove 11, into which the lower edge of the wall 2 of the Petri dish is locked. For this purpose, the diameter of the annular holding element 10 and the locking groove 11 are adapted to the cylindrical wall of the Petri dish.

Fig. 3b shows the carrier device 1 from below. Visible is the ring-shaped holding element 10, on the underside of which the laser-cuttable film 3

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ifglued. The bonding can be carried out using one of the methods described for Fig. 1 and Fig. 2.

Fig. 3c shows the ring-shaped holding element 10 in plan view with the locking groove 11 running around the top. The locking connection is liquid-tight, so that the laser-cuttable film 3 can be covered with liquid nutrient medium without liquid penetrating through the locking connection. At the same time, the locking connection forms a removable connection between the wall 2 of the Petri dish and the ring-shaped holding element 10 with the laser-cuttable film 3. This embodiment has the advantage that the ring-shaped holding element 10 with the laser-cuttable film 3 can be separated from the wall 2 of the Petri dish again if necessary.

Fig. 4 shows a device for laser microdissection with a carrier device 1 according to the invention. With the device for laser microdissection, a laser beam is moved over a specimen held relatively to it during cutting. The device for laser microdissection comprises a microscope 12 with a microscope stand 18 and a motor-driven xy table 13. The xy table 13 serves to hold the carrier device 1.

The carrier device 1 has a frame-shaped holder which is designed as the wall 2 of a Petri dish, which is typically made of plastic. The Petri dish has no bottom. The missing bottom of the Petri dish is replaced by a laser light-absorbing and thus laser-cuttable film 3 which is glued to the lower edge 4 of the wall 2. The glue is only applied in a thin layer and is therefore not shown. A biological living preparation 5 is applied or already grown on the laser light-absorbing and thus laser-cuttable film 3. In order to be able to illuminate the preparation 5 from below, the xy table 13 has a frame-shaped table opening 15.

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The microscope 12 shown is a transmitted light microscope. For this purpose, an illumination system 15 and a condenser 21 that illuminates the sample 4 are arranged under the xy table 13, and thus also under the specimen 5. At least one collecting container 29 for collecting the cut-out, interesting specimen area is arranged under the specimen 5. The light penetrating the specimen 5 reaches the objective 19 of the microscope 12. Inside the microscope 12, the light is guided via lenses and mirrors (not shown) to at least one eyepiece 22, through which an operator can view the specimen 5 arranged on the xy table 13.

A laser beam emanates from a laser 16, in this example a UV laser, which is coupled into an incident light illumination beam path with an optical axis 20. A laser scanning device 30 is arranged in the illumination beam path. The laser beam 17 passes through the laser scanning device 30 and reaches an objective 19 via an optical system 23, which focuses the laser beam 17 onto the specimen 5. The optical system 23 is preferably designed as a dichroic splitter, through which an imaging beam path emanating from the specimen 5 through the objective 19 reaches at least one eyepiece 22. Alternatively, the optical system 23 can consist of several optical components. This is the case, for example, if the laser beam 17 has to be deflected several times.

Furthermore, a diaphragm 24 is provided in the laser beam 17, with which the diameter of the laser beam 17 can be adjusted. The diaphragm 24 can be designed, for example, as a fixed diaphragm. In an advantageous embodiment, several fixed diaphragms can be arranged on a turret disk or a linear slide in order to introduce one of these fixed diaphragms into the beam path as the diaphragm 24 required in each case. The introduction into the laser beam 17 is carried out manually by the user or by motor.

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In this embodiment, the adjustment of the laser scanning device 30 and thus the adjustment of the laser beam 17 to the specimen 5 is carried out with a motor 31 assigned to the laser scanning device 30, a control unit 32 and a computer 26. The motor 31 is connected to the control unit 32, which supplies the control signals for controlling the motor 31. The control unit 32 is connected to the computer 26, to which a monitor 28 is connected. The image of the specimen 5 recorded by a camera 27 is shown on the monitor 28. The system consisting of computer 26, camera 27 and monitor 28 serves to observe and monitor the cutting process. The computer can thus send trigger signals to the laser to trigger laser pulses and to control the laser power, control the aperture motor 25 and control an autofocus device (not shown) for the laser 16. For this purpose, the computer 26 is connected to the laser 16 and supplies it with trigger signals for triggering laser pulses when a cutting process is carried out.

Using a computer mouse (not shown) or any other cursor control device, the sample area of interest of the preparation 5 to be cut out is circled on the monitor 28 using a mouse pointer. In this way, a desired target cutting line is defined on the monitor 28 in the camera image.

The laser scanning device 30 itself serves as a cutting line control unit which moves the laser beam 17 focused on the biological preparation 5 over the stationary preparation 5 during the cutting process. For this purpose, the xy table 13 is not moved horizontally, i.e. in the x-direction and in the y-direction, during the cutting process.

The laser beam 17 can be focused on the biological preparation 5 by manually adjusting the height of the xy table 13 while a user simultaneously visually controls the camera image. However, an embodiment of the device which includes an autofocus device (not shown) for the laser beam 17 is more user-friendly.

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By controlling the laser scanning device 30, the laser beam 17 can be guided to any position on the specimen 5. During the entire preparation of the section and also during the section itself, the biological specimen 5 remains alive, since the growth conditions in the carrier device 1 are always maintained.

By appropriately controlling the laser scanning device 30, the focused laser beam 17 is moved over the specimen 5, thereby creating a closed cutting line around the specimen area of interest. The specimen area of interest itself is never irradiated with the laser radiation, so that a damaging effect of the laser radiation on the specimen area of interest is excluded. After the cutting line is closed, the specimen area of interest is completely separated from the surrounding remaining specimen 5 and falls under the influence of gravity into the collecting container arranged underneath.

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List of reference symbols

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15

1. Carrier device

2. Wall of the Petri dish

3. laser cuttable film

4. lower edge of the wall

5. biological preparation

6. annular part

7. Opening

8. Adhesive film

9. Tab

10. ring-shaped retaining element

11. Locking groove

12. Microscope

13. movable xy-table

14. frame-shaped table opening

15. Lighting system

16. Laser

17. Laser beam

18. Microscope stand

19. Lens

20. optical axis

21. Condenser

22. Eyepiece

23. optical system

24. Aperture

25. Aperture motor

26. Calculator

27. Camera

28. Monitor

29. Collection container

30. Laser scanning device

31. Motor for laser scanning device

32. Control unit

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Claims (13)

1. A carrier device ( 1 ) for a biological preparation ( 5 ) which can be cut by means of laser microdissection and which is arranged on a freely stretched laser light absorbing film ( 3 ) which is applied to a frame-shaped holder, characterized in that the frame-shaped holder is essentially designed as a wall ( 2 ) of a Petri dish with a completely or partially missing bottom and that instead of the missing bottom only the laser light absorbing film ( 3 ) is arranged. 2. Carrier device ( 1 ) according to claim 1, characterized in that the entire bottom of the Petri dish is formed by the laser light-absorbing film ( 3 ). 3. Carrier device ( 1 ) according to claim 1, characterized in that the laser light-absorbing film ( 3 ) is a polyethylene naphthalate film (PEN). 4. Carrier device ( 1 ) according to claim 1, characterized in that the polyethylene naphthalate film has a thickness of 1.35 µm or 2.5 µm. 5. Carrier device ( 1 ) according to claim 1, characterized in that the laser light-absorbing film ( 3 ) is welded to the wall ( 2 ) of the Petri dish. 6. Carrier device ( 1 ) according to claim 1, characterized in that the wall ( 2 ) of the Petri dish is glued to the laser light-absorbing film ( 3 ). 7. Carrier device ( 1 ) according to claim 6, characterized in that the bonding by means of an adhesive film ( 8 ) designed in the form of a template is designed such that the adhesive film ( 8 ) is bonded on its one side to the cylindrical wall ( 2 ) and on its other side to the laser light-absorbing film ( 3 ). 8. Carrier device ( 1 ) according to claim 1, characterized in that the wall ( 2 ) of the Petri dish is cylindrical and that the film ( 3 ) is pre-prepared by means of welding or adhesive technology on an annular holding element ( 10 ), the diameter of which is adapted to the cylindrical wall ( 2 ) of the Petri dish and which has a locking groove ( 11 ) which allows the wall ( 2 ) of the Petri dish to lock into the annular holding element ( 10 ), so that a liquid-tight, detachable connection is created between the wall ( 2 ) of the Petri dish and the annular holding element ( 10 ) with the laser light-absorbing film ( 3 ). 9. Carrier device ( 1 ) according to one of the preceding claims, characterized in that the laser light-absorbing film ( 3 ) is designed to be hydrophilic. 10. Carrier device ( 1 ) according to one of the preceding claims, characterized in that the laser light-absorbing film ( 3 ) carries a nutrient medium for cell culture. 11. Carrier device ( 1 ) according to claim 10, characterized in that the nutrient medium is designed as a nutrient liquid. 12. Carrier device ( 1 ) according to claim 11, characterized in that the nutrient medium is designed as a nutrient gel. 13. Device for laser microdissection, - which comprises a microscope ( 12 ) with an objective ( 19 ) and an xy table ( 13 ) for receiving a biological preparation ( 5 ), - and a laser ( 16 ) whose laser beam ( 17 ) is coupled into an incident light illumination beam path, wherein the laser beam ( 17 ) is focused from above onto the biological preparation ( 5 ) through the objective ( 19 ) for cutting, - and a laser scanning device ( 30 ) which moves the laser beam ( 17 ) over the biological preparation ( 5 ) which is stationary during cutting, characterized in that the preparation ( 5 ) is a biological living cell culture which is arranged on a laser light-absorbing film ( 3 ) which is applied to a frame-shaped holder, wherein the frame-shaped holder is essentially designed as a wall ( 2 ) of a Petri dish and the laser light-absorbing film ( 3 ) is designed as the bottom of the Petri dish.