WO2011051738A1

WO2011051738A1 - Annealing incubator

Info

Publication number: WO2011051738A1
Application number: PCT/HU2010/000111
Authority: WO
Inventors: Csaba Niedetzky; János MATKÓ
Original assignee: Eötvös Lóránd Tudományegyetem
Priority date: 2009-10-30
Filing date: 2010-10-25
Publication date: 2011-05-05
Also published as: HU0900681D0; HUP0900681A2

Abstract

The invention relates to an annealing incubator (10) constructed for the inverted microscopic examination of biological preparations placed in a biological preparation plate (20). The invention is based on that it contains a nest (12) created for the plate, which nest (12) is formed by a base board (14), a cover board (16) and an internal wall (18) laterally surrounding the plate (20), in the lower and upper range of the plate (20) the internal wall (18) fits to the wall of the plate (20) with a clearance, while in the central range of the plate (20) it widens with a gap (22) with respect to the plate (20) allowing gas flow around the plate (20); the base board (14) contains an objective window (24) revealing the part of the plate (20) inside the nest (12) containing the biological preparation, while the cover board (16) contains an illumination window (26) revealing the part of the plate (20) inside the nest (12) containing the biological preparation, and the incubator (10) contains a gas-flow opening (28) for blowing annealing gas into the central wider range of the nest (12). The invention also relates to annealing equipment (50), which contains such an incubator (10), and to a procedure for manufacturing such incubators (10).

Description

ANNEALING INCUBATOR

The invention relates to an annealing incubator for the inverted microscopic examination of biological preparations placed in a biological preparation plate, especially in single- or multiple-well Microplate type plates.

The invention also relates to a procedure for manufacturing such annealing incubators and to annealing equipment containing such an incubator.

In the context of the present invention annealing means any temperature control (including heating, cooling, maintaining a given temperature). Nevertheless, in the case of biological preparations annealing means the setting and stable maintenance of temperatures typically higher than room temperature (maximum 52 °C), as in such fields of application generally it is not necessary to ensure temperatures lower than room temperature. At the same time, the annealing incubator and annealing equipment according to the invention also enables such applications.

When examining biological preparations it is important to keep the biological preparation at an appropriate - mostly stable - temperature, or, in a given case, the examination may be aimed at observing the behaviour of the biological preparation at given temperatures or in given temperature ranges, in which case the biological preparation may need to be exposed to several different temperatures. The equipment available in commercial distribution solve this task by placing the entire microscope in an annealing incubation chamber. This solution has several serious disadvantages:

1) the intricate and sensitive optical structure of the microscope is also exposed to the periodical thermal load involving heating and recooling and the resulting thermal expansion; 2) a relatively large space needs to be heated (or cooled), so the efficiency of annealing is not optimal from the aspect of energy consumption;

3) the preparation plates are difficult to change inside the incubation chamber, which hinders quick and efficient serial measurements.

The task to be solved with the invention is an annealing incubator and annealing equipment, which does not have the disadvantages of the state-of-the-art solutions.

Furthermore, the aim of the invention is to provide a procedure for manufacturing such incubators.

We recognised that if instead of the entire microscope only the biological preparation plate is heated, or cooled in a given case, then the optical structure of the microscope can be spared the heat load involving a change of temperature, and the efficiency of heating (or cooling) also improves, as no unnecessary space part are heated.

On the basis of the above recognitions, in accordance with the invention the set aim was solved with an annealing incubator constructed for the inverted microscopic examination of biological preparations placed in a biological preparation plate, which incubator contains a nest created for the plate, which nest is formed by a base board , a cover board and an internal wall laterally surrounding the plate, in the lower and upper range of the plate the internal wall fits to the wall of the plate with a clearance, while in the central range of the . plate it widens with a gap with respect to the plate allowing gas flow around the plate; the base board contains an objective window revealing the part of the plate inside the nest containing the biological preparation, while the cover board contains an illumination window revealing the part of the plate inside the nest containing the biological preparation, and the incubator contains a gas-flow opening for blowing annealing gas into the central wider range of the nest. 10 000111

3

Favourably, between the base board and the cover board the incubator contains further intermediate boards, in which there are openings forming the nest, in such a way that the internal edge of the opening of the boards forms the internal wall of the nest. So basically the incubator consists of boards placed on top of each other, in which the nest accommodating the preparation plate is formed by the openings cut into the individual boards altogether.

Favourably the cover board slides on the top intermediate board between a nest closing and a net opening position. In the nest closing position the illumination window is situated above the nest, while in the nest opening position the cover board is removed from above the nest. The preparation plate can be removed from or placed inside the nest when the cover board is in the nest opening position.

Favourably in the plane of the cover board there is a stop plate attached to one side of the top intermediate board, and in the nest closing position the cover board hits the stop plate. Favourably there is a fastening plate attached to the stop plate on the side opposite the top intermediate board, and the size and shape of this fastening plate is chosen to ensure that in the nest closing position of the cover board the fastening plate extends partly above the cover board holding down the cover board.

Favourably the external wall of the incubator is constructed to suit the geometry of standard microtitre plates. Suiting the shape of the microtitre plates standardised in laboratory practice is important, because by this it is ensured that the annealing incubator can be used on any type of inverted microscopes, with a stage of any make (either manual or automatic), as generally adapters for standard microtitre plates are included in the product selection of all manufacturers.

Favourably the nest of the incubator is dimensioned to be able to accommodate 2-, 4- or 8-well Microplate type plates, for example explicitly suitable for the size of Lab-Tek Micro Plate types, which are the most widely used in microscope technology.

The invention also relates to annealing equipment using the incubator described above, which is based on that it contains a gas annealing module, the output of which is connected to the gas-flow opening of the incubator via a gas pipe.

The invention also relates to a procedure for manufacturing annealing incubators for the inverted microscopic examination of biological preparations placed in a biological preparation plate, which incubator contains a nest accommodating the plate. The procedure is based on that:

- openings of different sizes are cut into boards, so that one of the boards - the base board - contains an objective window revealing the part of the plate inside the nest containing the biological preparation, while another board - the cover board - contains an illumination window revealing the part of the plate inside the nest containing the biological preparation, and the intermediate boards have increasing and decreasing opening sizes,

- the base board and the intermediate boards are attached on top of each other so that the size of their opening increases and then decreases,

- through one or more boards forming the wall of the nest a gas- flow opening is created.

Favourably the cover board is fixed onto the top intermediate board with a releasable connection enabling the opening and closing of the nest. Releasable connection between the cover board ad the top intermediate board means all types of connections, which make it possible to remove the cover board from above the opening of the top intermediate board allowing by this to place the preparation plate inside the nest. Favourably during the procedure the following operations are performed:

- a hole is drilled into at least two of the corners of the boards,

- a guide pin is inserted through the holes of the base board, - one of the intermediate boards is pulled onto the guide pins through its holes, and adhesive is applied on at least one of the two sides of the intermediate board and the base board facing each other,

-. the intermediate board and the base board are pressed together, and

- the placement and gluing of the board is repeated with a further intermediate board.

Favourably plastic non-transparent black Plexiglas boards are used, and the gas-flow opening is created with the help of a milling machine after fixing the boards to each other.

Further details of the invention are described on the basis of construction examples, with the help of a drawing. In the drawing

figure 1 is the perspective view of a favourable construction of the incubator according to the invention,

figure 2 is the top view of the base board of the incubator according to the invention,

figure 3 is the top view of the first intermediate board of the incubator,

figure 4 is the top view of the second intermediate board of the incubator,

figure 5 is the top view of the third intermediate board of the incubator,

figure 6 is the top view of the cover board of the incubator, figure 7 is the top view of the stop plate of the incubator, figure 8 is the top view of the fastening plate of the incubator, figure 9 is the block diagram of the annealing equipment according to the invention.

Figure 1 shows a favourable construction of the incubator 10 according to the invention. The incubator 10 has a nest 12, which delimited by the base board 14 at the bottom, by the cover board 16 at the top, and by the internal wall 18 of the incubator 10 on the sides. The size and geometry of the nest 12 is chosen so that from the direction of the top of the incubator 10 - in a given case after opening the cover board 16 - the biological preparation plate 20 can be placed inside it. Favourably the nest 12 of the annealing incubator 10 can be dimensioned to make it suitable for accommodating single- or multiple- well Microplate (especially a 2-, 4- or 8-well Lab-Tek Microplate) type plates 20.

The nest 12 and the internal wall 18 of the incubator is constructed in such a way that along the lower and upper range of the inserted preparation holder 20 they rest closely against the plate 20, while along the central range of the plate 20 an appropriate gap 22 is left between the plate 20 and the internal wall 8 for allowing the flowing of the annealing gas (see figure 4).

The incubator 10 is constructed for inverted microscopic examinations, the microscope objective is situated below the stage, while the object is illuminated from above. In order to illuminate the biological preparation and observe it through the microscope, in the base board 14 and cover board 16 of the incubator 10 there is an objective window 24 and an illumination window 26. The objective window 24 in the base board 14 reveals the part of the plate 20 inserted in the nest 12 containing the biological preparation, and so the biological preparation can be studied from below through the microscope objective. The illumination window in the cover board 16 makes it possible to transilluminate the biological preparation in the inverted microscope from above with a light beam (e.g. laser beam). In the nest 12 of the incubator 10 the biological preparation is annealed by entering heated (or cooled) gas, e.g. air. For this purpose the incubator 10 has a gas-flow opening 28 leading into the central wider range of the nest 12, that is into the gap 22 between the inserted plate 20 and the internal wall 18 of the incubator 10.

Figures 2-8 show the building units of a construction example constructed from parallel boards 30 placed on top of each other: the base board 14 is at the bottom (figure 2), it is followed by a first, second and third intermediate board 32 (figure 3, 4 and 5), and finally by the cover board 16 (figure 6). There is also a stop plate 34 situated in the same plane as the cover board 16 (figure 7), and the fastening plate 36 is attached to it from above (figure 8).

Favourably the boards 30 are made of a few mm thick, non- transparent (to prevent the light illuminating the preparation from advancing in it), black (to reduce harmful light reflections) Plexiglas boards, and favourably they are attached to each other with an adhesive. Favourably the base board 14 is thinner (e.g. 1-1.5 mm thick), while the other boards 30 are thicker (about 4-6 mm thick, e.g. 5 mm thick). Favourably the boards 30 are non-transparent, so the sample, that is the biological preparation is illuminated exclusively by the light beam arriving through the window 26 of the cover board 16.

When illustrating the boards 30, we marked the place of the biological preparation plate 20 with a dotted line. It can be seen that the window 24 cut into the base board 14 from the microscope objective is smaller than the bottom of the plate 20 (e.g. a Microplate type plate), so it rests against the range of the base board 14 around the window 24. Favourably the base board 14 is made of a thin (e.g. 1-1.5 mm thick) Plexiglas board so that the examined biological preparation is not lifted from the microscope stage too much, which would make it difficult, or practically impossible above a certain distance, to focus on the sample. For this reason the base board 14 can even be significantly thinner than 1 mm. Also upon optical considerations - so that there are fewer transitions between media in the way of the light beam - the window 24 can be left empty. However, it is also possible to cover the window 24 with glass or with some other plate less disturbing from an optical aspect, in order to protect the sensitive microscope objective from the heated gas flowing in the gap 22.

The internal wall 18 of the incubator 10, and also the nest 12, is formed by the internal edges 40 of the openings 38 of the intermediate boards 32. It can be seen that the intermediate boards 32 have openings 38 of different sizes. The preparation plate 20 fits with a clearance into the opening 38 of the first intermediate board 32 neighbouring the base board 14, while there is a significant gap 22 left between the internal edges 40 of the opening 38 of the second intermediate board 32 falling in the central range of the plate 20 and the plate 20, in which gap 22 the annealing gas can flow freely, ensuring by this appropriate annealing along the entire periphery of the plate 20. Favourably the opening 38 of the third intermediate board 32 accommodates the plate 20 inserted in the nest 12 with a clearance again, so the annealing gas cannot leave the gap 22 too fast, which increases the efficiency of annealing. It is also possible to cut the openings 38 in the first and third intermediate boards 32 diagonally, rather than with a perpendicular edge, as a result of which the cross- section of the opening 38 gradually narrower towards the bottom of the first intermediate board 32 and the top of the third intermediate board 32. It may be favourable to make indents in the edge 40 of the opening 38 of the third intermediate board 32, on two sides, to make it easier to grab the preparation plate 20 inserted in the nest 12 and lift it out with one's fingers or using a tool.

Favourably the intermediate boards 32 are 4-6 mm thick, and favourable they are cut out from non-transparent Plexiglas boards. The external geometry of the boards 30 is chosen to follow the shape of the microtitre plates regarded as standard in laboratory practice. Thus the incubator 10 can be used with any type of inverted microscope, with a stage of any make (manual or automatic), as generally adapters for standard microtitre plates are included in the product selection of all manufacturers. Such adapters assume that a corner on the right or on the left is cut off, so favourably - to make the incubator 10 compatible both with right-side and left-side adapters - cut corners 42 are created on the boards 30 at the necessary height both on the right side and on the left side. In the present case such corners were created at two corners of the second and third intermediate boards 32 and the cover board 16. Obviously, depending on the microscope stage and the adapter, a different external geometry can also be realised through the appropriate construction of the boards 30 of the incubator 10 to enable fastening on the given stage or in the given adapter.

Favourably the cover board 16 is attached to the third intermediate board 32 via a releasable connection, In the case of the present construction example, the releasable connection is realised with the help of the stop plate 34 and the fastening plate 36. Practically the stop plate 34 attached to the third intermediate board functions as a distance piece for the fastening plate 36, the arresting range 44 of which extends over the stop plate 34. The stop plate 34 keeps the fastening plate 36 away from the third intermediate board 32 exactly at a distance that makes it possible to slide in the cover board 16 between the arresting range 44 of the fastening plate 36 and the third intermediate board 32, where it is arrested by the fastening plate 36 without any clearance, ensuring by this the releasable connection, for the releasing of which the cover board 16 is pulled out from below the fastening plate 36 from the same direction. The sides of the cover board 16 and the stop plate 34 contacting each other are constructed in a complementary way, so that when the cover board 16 is pushed below the arresting range 44 of the fastening plate 36 until it hits the stop plate 34, favourably the cover board 16 cannot move laterally either. In the case of the present construction example it is solved with extension pieces 46 extending from the cover board 16 towards the stop plate 34, closely fitting the opposing periphery of the stop plate 34 created in a complementary way. Obviously numerous other versions of the complementary forms preventing lateral movement can be realised too.

Apart from the example above, the releasable connection between the cover board 16 and the third (top) intermediate board 32 can mean all connections, which make it possible to remove the cover board 16 from above the opening 38 of the third (top) intermediate board 32 in order to free the opening 38 and place the preparation plate 20 in the nest 12 freely. For example the cover board 16 can also be attached to the third intermediate board 32 (or to any lower intermediate board 32) with a pivoted joint, releasing the connection simply means that the cover board 16 is folded out around the picot, in the course of which the cover board 16 is lifted away from the upper surface of the third intermediate board 32 leaving the opening 38 of the intermediate plate 32, that is the entrance to the nest 12 free. Completely different types of connections, such as form-locking complementary, clicking, magnetic, etc. connections are also possible, which are suitable for releasing the cover board 16 from the intermediate board 32 at least to an extent to make its opening 38 free so as to insert the plate 20. Obviously it is necessary, if the cross-section of the window 26 of the cover board 16 is smaller than the plate 20.

The window 26 of the cover board 16 is dimensioned to enable the transillumination of the biological preparation during the inverted microscopic examination on the one part, and on the other part to allow the annealing gas entered into the central wider range of the nest 12 to leave. The latter takes place automatically, if the plate 20 is a commonly used 2-, 4- or 8-well Microplate, in which case the annealing gas reaches the window 26 through the gaps between the individual wells of the plate 20 too. Obviously it is not necessary to discharge the annealing gas through the window 26 of the cover board 16, in a given case the window 26 can be covered with a light-permeable plate (for example glass plate) to protect the biological preparation from slight contaminations coming from above. In this case a gas outlet opening can be created somewhere else.

Favourably the cover board 16 locks the plate 20 inside the nest 12, so favourably the cross-section of the window 26 is smaller than the cross-section of the plate 20, so the plate 20 can be removed from the nest 12 only after releasing the releasable connection of the cover board 6 and removing the cover board 6 at least partially.

The annealing incubator 10 according to the invention is assembled as described below, using the boards 30 shown in figures 2- 8. It can be seen that there are holes 48 drilled into the corners of the base board 14, the three intermediate boards 32, and into the corners of the stop plate 34 and the fastening plate 36 on the one side. A guide pin is placed in each one of the four holes 48 drilled into the base board, then the intermediate boards 32 are stringed onto these guide pins one after the other. Favourably adhesive should be applied on at least one of the contacting surfaces of the boards 30. After putting the individual boards 30 in their place, the boards are pressed together to allow the adhesive to spread appropriately. After the placement of the third intermediate board 32, the stop plate 34 and the fastening plate 36 are also stringed onto the guide pins on the appropriate side. Obviously the two holes and guide pins on the stop plate 34 are also enough for performing the above operation, as precise gluing can be realised after fixing the given planes in two points.

Favourably the stringed and glued boards 30 are kept pressed together for a certain period, for example in the case of Plexiglas boards and Plexiglas glue they are gripped in a vice for about an hour to allow the glue to diffuse into the Plexiglas boards 30, in order to obtain a massive block enclosed on the sides. After this the guide pins can be removed. However, favourably threaded spindles (headless threaded rods) of a length equivalent to the height of the block to be created are used as guide pins, the ends of which at the top and at the bottom exactly coincide with the external plane of the cover board 16 and the base board 14, and these metal pins are left in the holes 48. In the case of another favourable construction bolts can be used as guide. pins, and by placing nuts on the protruding end of the bolts, with the help of the nuts the boards 30 can be fastened together even more tightly.

The cover board 16 is fixed to the intermediate board 32 on top with a releasable connection, for example in the present case by sliding the cover board 6 below the fastening plate 36.

It can be seen that the boards 30 shown in figures 2-8 do have a lateral opening for entering gas. The reason for this is that favourably the gas-flow opening 28 of the incubator 10 is created subsequently, after the individual boards 30 have been attached to each other. In the present case the gas-flow opening 28 can be created favourably with the help of a milling machine in one of the side walls of the Plexiglas block created by gluing, so that the opening 28 leads into the central wider range of the nest 12. By this we also mean constructions, where the gas-flow opening does not simply enter the central wider range of the nest 12, but it also has a larger cross-section than that, so it also reaches into the narrower lower and higher ranges of the nest 12 (in the present case in the range of the first and third intermediate board 32) ensuring gas flow directly into those ranges too.

Favourably the opening 28 is created before fitting the cover board 16 in its place, and favourably on the side of the incubator 10 where the stop plate 34 and the fastening plate 36 are fixed permanently. In the case of the present construction example the place of the gas-flow opening 28 to be created and the direction of the gas flow is marked with arrow A in figures 2-8.

Figure 10 shows the block diagram of annealing equipment 50 containing an annealing incubator 10 according to the invention. The gas-glow opening 28 of the incubator 10 is connected to a gas annealing (heating or - in a given case - cooling) module 52 via a gas pipe 54. The gas annealing module 52 heats (or in a given case cools) the input gas, for example air, which then flows through the gas pipe 54 and the gas-flow opening 28 of the incubator 10 and enters the nest 12, in the wider central range of which (practically in the gap 22) it flows around the plate 20 and anneals (heats or, in a given case, cools) the biological preparation stored therein.

The gas annealing module 52 can be, for example, a heat exchanger, which has, for example, built-in active current generator heating elements. The temperature of the latter is controlled with temperature controller 55, for example a Physiological-Biological Temperature Controller distributed by Supertech Instruments.

One or more heat sensing devices 56 are used for controlling the temperature. For example a high-precision Dallas Semiconductor DS18S20 thermometer can be placed in the gas annealing (heat exchanger) module 52. By measuring the temperature in the heat exchanger module 52 great fluctuations deriving from temperature regulation can be avoided, that is temperature control can be realised with a small hysteresis. At the same time the average temperature value will be less precise, the gas temperature inside the heat exchanger module 52 can be different from the temperature reached in the nest 12 of the incubator 10 by as much as about 10 °C, because some heat loss occurs inevitably along the gas pipe 54.

On the one part the fault deriving from this can be eliminated by calibration when the annealing equipment is put into operation. On the other part, in order to control the temperature a further thermometer 56 can be placed in the incubator 10, for example at the point in the internal wall 18 of the nest 12 opposite the gas-flow opening 28, so it measures directly the temperature inside the nest 12. For example in the case of the above construction example, at the edge 40 of the opening 38 of the second intermediate board 32 opposite the gas-flow opening 28 a thermometer 56 can be placed, the signal transmitting cable of which can be taken to the temperature controller 55 through the hole 58 leading into the internal edge 40 of the intermediate board 32. A suitable thermometer 56 can be for example a precision temperature measuring circuit operating with a Pt100 sensor, which Pt100 sensor is arranged in or at the internal wall 18 of the nest 12.

Favourable the gas pipe 54 connecting the module 52 with the gas-flow opening 28 of the incubator 10 is a flexible, heat-insulated, plastic pipe. Favourably the heat exchanger module 52 can be placed relatively close to the incubator 10, for example on an adjustable stand extending over the microscope, and by this the length of the gas pipe 54 can be minimised, and so the intermediate heat loss can be reduced.

Filtered air of room temperature and a constant volume flow rate produced by a gas source 60, for example by an Air Flow Heater Controller manufactured by Supertech Instruments, is taken to the input of the heat exchanger module 52 favourably through a flexible plastic pipe. When choosing the volume flow rate it must be taken into consideration that if the volume flow rate is too low, it will not result in satisfactory annealing, while if the volume flow rate is too high, it may result in the mechanical vibration of the preparation during measuring. We found that air output of a volume flow rate of about 2-20 l/minute is suitable for annealing the biological preparation placed in the nest 12 of the incubator 10. Favourably the Air Flow Heater Controller serving as a gas source 60 contains a thermometer 56, for example a precision temperature measuring circuit operating with a Pt100 sensor. Favourably the Pt 00 sensor is situated inside the incubator 10, on the side of the nest 12 opposite the incoming air flow, as it is described above in connection with the temperature controller 55. In this way, independently from handling the air with a temperature regulated I the heat exchanger module 52 by the temperature controller (e.g. Physiological-Biological Temperature Controller), the actual current temperature of the inside of the annealing vessel is always displayed on the LSD screen placed on the front of the Air Flow Heater Controller serving as a gas source 60.

The incubator 10 constructed from boards 30 described here is only an example, the main advantages of the invention are represented by the nest 12 suitable for annealing constructed for the preparation plate 20 and its special geometry, which can be realised differently too, as it is obvious for a person skilled in the art, for example an incubator 10 of a similar shape as the above can be made by injection-moulding, in the course of which two separate elements - the block accommodating the nest 12 and the cover board 6 attached to it with a releasable connection - are manufactured.

The use of Plexiglas boards 30 also serves as an example, they are advantageous first of all because they are cost efficient and favourable workable. At the same time, for example the base board 14 can also be made of a different heat insulating material, for example Bakelite, in the case of which the desired thinness of even less than 1 mm is easier to ensure.

Obviously a person skilled in the art can think of alternative solutions other than the construction examples described herein, which are within the scope of protection defined by the claims.

Claims

1. Annealing incubator (10) constructed for the inverted microscopic examination of biological preparations placed in a biological preparation plate (20), characterised by that it contains a nest (12) created for the plate, which nest (12) is formed by a base board (14), a cover board (16) and an internal wall (18) laterally surrounding the plate (20), in the lower and upper range of the plate (20) the internal wall (18) fits to the wall of the plate (20) with a clearance, while in the central range of the plate (20) it widens with a gap (22) with respect to the plate (20) allowing gas flow around the plate (20); the base board (14) contains an objective window (24) revealing the part of the plate (20) inside the nest (12) containing the biological preparation, while the cover board (16) contains an illumination window (26) revealing the part of the plate (20) inside the nest (12) containing the biological preparation, and the incubator (10) contains a gas-flow opening (28) for blowing annealing gas into the central wider range of the nest (12).

2. Incubator as in claim 1 , characterised by that between the base board (14) and the cover board (16) it contains further intermediate boards (32), in which there are openings (38) forming the nest (12), in such a way that the internal edge (40) of the opening (38) of the intermediate boards (32) forms the internal wall (18) of the nest (12).

3. Incubator as in claim 2, characterised by that the cover board (16) slides on the top intermediate board (32) between a nest closing and a net opening position, and in the nest closing position the illumination window (26) is situated above the nest (12), while in the nest opening position the cover board (16) is removed from above the nest (12).

4. Incubator as in claim 3, characterised by that in the plane of the cover board (16) there is a stop plate (34) attached to one side of the top intermediate board (32), and in the nest closing position the cover board ( 6) hits the stop plate (34), and there is a fastening plate (36) attached to the stop plate (34) on the side opposite the top intermediate board (32), and the size and shape of this fastening plate (36) is chosen to ensure that in the nest closing position of the cover board (16) the fastening plate (36) extends partly above the cover board (16) holding down the cover board ( 6).

5. Incubator as in any of claims 1-4, characterised by that its external wall is constructed to suit the geometry of standard microtitre plates.

6. Incubator as in any of claims 1-5, characterised by that the nest is dimensioned to be able to accommodate 2-, 4- or 8-well Microplate type plates.

7. Annealing equipment (50), characterised by that it contains an incubator (10) as in any of claims 1-6, and it contains a gas annealing module (52), the output of which is connected to the gas-flow opening (28) of the incubator (10) via a gas pipe (54).

8. Procedure for manufacturing annealing incubators for the inverted microscopic examination of biological preparations placed in a biological preparation plate, which incubator contains a nest accommodating the plate characterised by that

- openings of different sizes are cut into boards, so that one of the boards - the base board - contains an objective window revealing the part of the plate inside the nest containing the biological preparation, while another board - the cover board - contains an illumination window revealing the part of the plate inside the nest containing the biological preparation, and the intermediate boards have increasing and decreasing opening sizes; - the base board and the intermediate boards are attached on top of each other starting with base board, so that the size of their opening increases and then decreases;

9. Procedure as in claim 8, characterised by that the cover board is fixed onto the top intermediate board with a releasable connection enabling the opening and closing of the nest.

10. Procedure as in claim 8 or 9, characterised by that

- a hole is drilled into at least two of the corners of the boards,

- a guide pin is inserted through the holes of the base board,

- one of the intermediate boards is pulled onto the guide pins through its holes, and adhesive is applied on at least one of the two sides of the intermediate board and the base board facing each other,

- the intermediate board and the base board are pressed together, and

1 1. Procedure as in claim 8-10, characterised by that plastic, especially Plexiglas boards are used, and the gas-flow opening is created with the help of a milling machine after fixing the boards to each other.