Description

The invention refers to a holding device for microscopy application, wherein the holding device enables a cell culture insert to be positioned under a microscope, wherein the insert has a membrane which is configured to be provided with the biological sample and which is surrounded by a sidewall, and a microscopy chamber.

Biological samples such as tissue sections of biological samples are well used in the field of natural sciences for biological, chemical or physical examinations, for instance for electrophysiological examinations. In general, the tissue sections can be investigated directly after acute slice preparation or they can be cultivated prior to investigation in special inserts such as Millicel® Organotypic Cell Culture Inserts over a long period of time. Comparable culture inserts are known from US 5,652,142, US 5,578,492 and US 5,468,638. The inserts have a membrane which is configured to be provided with the tissue section and which is surrounded by a frame, in particular by a sidewall. In order to ensure access of the nutrient solution to the tissue during incubation the inserts have foot-like projections elevating the membrane over the bottom of the incubation device.

During the incubation period, the tissue section forms a firm mechanical connection with the membrane. For immediate

investigation of freshly prepared samples, attachment of the tissue to the membrane can be achieved using biocompatible adhesives, e.g. Poly-D-Lysine . In order to examine the tissue sections under a microscope, a membrane portion carrying the tissue section is cut out and placed on a microscope table via a tweezer. However, it has been shown that this preparation procedure stresses the tissue section as mechanical loads such as bending and/or pressure forces are Introduced inevitably into the tissue section.

Further on, in general the cut tissue section is exposed to a fluid such as a buffer solution or nutrient solution during examinations. In order to fix the cut tissue section in the solution under the microscope, the tissue section is fixed by so called „ Slice-Harps" -fixtures comprising a metal ring which is covered with nylon threads. It has been shown that this fixation procedure also stresses the tissue section as

mechanical loads are introduced inevitably into the tissue section. In addition, the nylon threads can cut into the tissue section which can lead to an irreversible tissue damage.

However, a vibration free and drift free fixation of the tissue section is essential for microscope examinations.

It is therefore an object of the present invention to provide an improved holding device and a microscopy chamber in order to overcome the above-mentioned disadvantages, in particular to enable a stress- free placement and fixation of a biological sample, for instance a tissue section, under a microscope for examinations.

The object is solved by a holding device with the features of claim 1 and by a microscopy chamber with the features of claim 12,

According to the invention, a holding device for holding a cell culture insert for microscopy applications, wherein the insert has a membrane which is configured to be provided with the biological sample and which is surrounded by a frame, comprises at least a base plate and a cover. The base plate is configured to carry the insert, wherein the cover is configured to fix the insert on the base plate. In a closed state of the holding device, the cover interacts with the base plate and produces a tension force acting on the membrane of the insert.

A microscopy chamber according to the invention has an

integrated inventive holding device.

The holding device enables the biological sample such as a tissue section to stay in the insert during microscope

examinations, and even more, to be placed in a fixed and stress-free manner under the microscope. Contrary to the known prior art, the tissue section has not to be cut out of the insert membrane and/or hold in place under the microscope via a Slice Harp. In consequence, the holding device according to the invention prevents the tissue section from placement stress and fixation stress. Additionally, due to the tensioning of the membrane in the fixed position, spontaneous displacements of the membrane are effectively prevented. Thus, the use of the inventive holding device leads to results which are more preciously compared with examinations based on the known placement and fixation procedures. Moreover, the integrity of the frame structure and the membrane of the cell culture insert is preserved throughout the examination in the holding device under a microscope. Therefore, tissue slices can be reincubated for additional investigations. Intact cell culture inserts can also be re-used after cleaning. Beside tissue sections, further examples of biological samples are 2D and 3D animal or plant cell cultures, Nematodes, or larvae to be examined .

According to a preferred embodiment, in an opened state of the holding device the insert rests on its membrane. In the preferred embodiment, the membrane rests on a bottom of the base plate. By means of this, a large supporting surface is established. However, it is also possible to deposit the membrane on at least one projection being outside of the bottom, in the latter case, it is preferred if the at least one projection has a form that correspondence to an outer

circumference of the membrane. This enables a uniform

tensioning of the membrane over its circumference . For

instance, if the membrane has a circumference of a circle, the projection preferably is an annular ring with an outer diameter which is equal or almost equal to an outer diameter of the membrane .

In order to produce a defined tension force without damaging the membrane in the closed state of the holding device, the frame of the insert can be clamped between the base plate and the cover .

Preferably, in the closed state the base plate and the cover guide the membrane via opposite diverting means thereby tensioning the membrane. By means of this, a smooth guidance of the membrane is realised by an annular ring and a damage of the membrane is prevented.

In order to reduce the height of the holding device, the base plate can have a recess for creating space for each insert foot. By means of this, the height of the holding device can be reduced, for instance.

A misalignment of the cover in the closed state can be

prevented if the cover is held in place via a closing force. Preferably, the closing force enables a quick and easy opening and closing of the holding device and a reliable locking without avoiding any negative impact on the biological sample to be examined. In one embodiment, the closing force is a magnetic force. Therefore, the base plate and the cover can be provided with corresponding magnets, for instance so-called Neodymium magnets.

Preferably, the base plate forms a fluid-tight microscope chamber configured to receive the insert . By means of this, the biological sample can be examined in an optimal environment as it can be surrounded by a fluid such as a buffer solution or a nutrient solution during examinations. In order to enable the examinations , a bottom of the chamber consists of a transparent material such as glass which enables light to pass through. On its top side, the chamber is preferably opened to provide access for water immersion objective lenses and additional experimental devices .

In one embodiment , the cover is provided with fluid ports and/or connections for measuring means . The fluid ports enable the holding device to be incorporated in fluid systems such as a buffer solution system, a nutrient solution system and the like, so that an optimal environment can be established for the biological sample during examinations. The at least one connection enable the holding device to install a sensor such as a temperature sensor, for instance for controlling the temperature of a solution surrounding the biological sample . It should be noted that the term "connection" also means a mechanical mount for a measuring means such a sensor .

Preferably, the cover has a sickle-shaped heating element, preferably an annular heating element configured to be

positioned inside the insert . By means of the sickle shape , the heating element surrounds the biological sample at least partially. By means of an annular shape , the heating element surrounds completely or almost completely the biological sample . Any negative impact of the objective lens that touches the fluid surface and that acts as heat sink are overcome . In consequence, via the annular heating element , the buffer fluid surrounding the biological sample can be heated to a required temperature . As the heating takes place near the biological sample, and the temperature sensor is mounted in a defined position the buffer temperature can be reproducibly adjusted very preciously. An overheating of the buffer fluid to a temperature which is higher than an allowable maximum

temperature, for instance 35°C to 37°C, is prevented . In addition, an external preheating device for the buffer solution to be applied to the biological sample can be omitted so that the solution can enter the chamber by room temperature which is about 25°C, As a consequence, overheating- induced outgassing of gas-saturated buffers is omitted, therefore gas-saturation of the buffer solution is optimal when being applied to the biological sample .

In one embodiment , the heating element has a hollow structure configured to be incorporated in a hot fluid system. The main advantage of a fluid-based heating element is that compared with an electrical heating element, electrical or

electromagnetic effects do not exist as no current runs through the heating element , neither close nor far away from the biological sample. Thus , conducting electrophysiological examinations , measuring results having a high quality are obtained. Preferred hot fluids are water and oil . However, also a preheated gas can be used. However, an electrical ring as heating element is still an option. The use of the adequate heating element depends mainly on the specific examination .

In order to have unobstructed access to the biological sample in the insert , the ports for the at least one fluid system and/or measuring sensors are positioned outside an access area for the objective lens and additional devices . By means of this, the cover has at least one section for ports only and at least one section for handling during the examination.

Further advantageous embodiments are disclosed in the dependent claims, the description and the figures .

In the following, a preferred embodiment of the present invention is explained with respect to accompanying drawings .

As is to be understood, the various elements and components are depicted as examples only , may be facultative and/or combined in a manner different than that depicted. Reference signs for related elements are used comprehensively and not defined again for each figure . Shown is schematically in Figure 1 a perspective top view of an exemplary known cell culture insert for carrying a biological sample to be examined under a microscope,

Figure 2 a perspective bottom view of the insert,

Figure 3 a top view of a preferred embodiment of the holding device according to the invention,

Figure 4 a bottom view of the holding device,

Figure 5 an exploded view of the holding device,

Figure 6 a sectional view of the holding device in an opened state ,

Figure 7 a sectional view of the holding device in a closed state,

Figure 8 a perspective single view of a base plate of the

inventive holding device,

Figure 9 a sectional view of the base plate,

Figure 10a a perspective top view of a cover of the inventive holding device,

Figure 10b a perspective bottom view of the cover,

Figure 11 a further bottom view of the cover indicating the heating element as well as the buffer inlet and outlet channel,

Figure 12 a sectional view of the cover,

Figure 13 a top view of the cover indicating the heating

element as well as the buffer inlet and outlet channel ,

Figure 14 a perspective sectional view of the holding device and the insert showing the position of the heating element in the closed position of the holding device,

Figure 15 a sectional view of the heating element of the cover, Figure 16 a sectional view of the holding device in the opened state, and

Figure 17 a sectional view of the holding device in the closed state.

In figure . and 2 an exemplary cell culture insert 1 used for carrying a biological sample 3. In the shown embodiment , the biological sample is a tissue section 3. The shown cell culture insert 1 , in following called insert 1, is well known as

Millicel® Organotypic Cell Culture Insert, It comprises a membrane 2, a sidewall 4 and feet 6. The membrane 2 has circular shape and on its upper side 8 it is adapted to be provided with the tissue section 3 to be examined. Exemplary examinations are electrophysiological examinations under a microscope, The sidewall 4 surrounds the membrane 2 and draws a frame around the membrane 2 , The feet 6 extend from the sidewall 4 opposite to the upper surface 8 of the membrane 2 and keeps the membrane 2 in a distance to a ground if the insert is placed in a breeding device or on a working table and the like, such that a direct contact between a bottom surface 10 of the membrane 2 and the ground is prevented. Here, the known insert 1 has three feet 6 which are placed uniformly to each other. In general, the membrane 2 is a kind of material mesh and the frame 4 is made of a rigid material .

In figures 3, 4 and 5, a preferred embodiment of a holding device 12 is shown in different views. In figures 3 and 4, the holding device 12 is shown in its closed state from the top and from the bottom, respectively. In figure 5, the holding device 12 is shown in its opened state with its essential parts 14, 16 separately. The essential parts of the holding device 12 are a base plate 14 and a cover 16,

The base plate 14 has a rectangular shape with a large

extension in longitudinal direction x and a short extension in transverse direction y . On its longitudinal sides retainment elements 18 for engaging the holding device 12 in corresponding microscope mounts are provided. In the shown embodiment four slot like retainment elements 18 are pairwise provided on the opposite longitudinal sides . It should be noted that the retainment elements 18 can be adapted freely to every

microscope mounts . In a centre portion of the base plate 14 an annular opening 20 is provided for receiving the insert 1, The bottom of the opening 20 is closed by a transparent disc 22,

The cover 16 has a rectangular shape with a large extension in longitudinal direction x and a short extension in transverse direction y. For lifting the cover 16 from the base plate 14 and for depositing the cover 16 on the base plate 14, the cover 16 has a plate like handle 30 on one of its short transverse sides, In a centre portion 54 ( s . figures 10b and 12) of the cover 16, a hole 32 is provided.

In the closed state, in order to avoid a misalignment in longitudinal direction x or transverse direction y, the cover 16 is positioned in a recess 34 of the base plate 14 having a form that corresponds to the outer rectangular form of the cover 16. In order to avoid an unintended loss of the cover 16 in the closed state, not shown magnets are positioned in corresponding recesses 36, 38 of the base plate 14 and of the cover 16 producing a magnetic closing force. Here, three magnets are positioned in each corner of the recess 34 in the base plate 14 and in opposite corner areas of the cover 16 ,

As illustrated igure 6, in the opened state of the holding device 12 the cover 16 is lifted up from the base plate 14. The insert 1 is placed in the centre portion of the base plate 14 in the opening 20. The membrane 2 basically deposits on the transparent disc 22, wherein the frame 4 of the insert 1 with its feet 6 is in a distance to opposite base plate areas. Thus, the opened state the insert 1 rests solely on its membrane 2.

As illustrated in figure 7, in the closed state of the holding device 12 the insert 1 is fixed between the base plate 14 and the cover 16. In particular, a vertical closing force Fc produced by the magnets is acting on the cover 16. The frame 4 is moved down and clamped between the base plate 14 and the cover 16. Due to the downward movement of the frame 4, a horizontal tension force Ft is acting on the membrane 2 such that the membrane 2 is tensioned over its circumference radially.

In figures 8 and 9, a more detailed explanation of the base plate 14 follows. Figure 8 illustrates a top view and figure 12 represents a sectional view along line AA in figure 8. As mentioned before, the base plate has a rectangular shape. It is made of a rigid material. Preferably it is produced via additive manufacturing such as laser sintering or 3D-printing . In order to avoid any drawback on the tissue section, the base plate 14 is made of a biocompatible material, in particular a 3D-printable biocompatible polymeric material. A preferred 3D- printing method is stereolithography.

The opening 20 in the centre portion has a circular shape and is closed at its bottom side via the transparent disc 22. The transparent disc 22 has a constant thickness and is for instance a separately produced glass bottom and in a fluid- tight engagement with a contact area 24 of the base plate 14,

By means of this, it is guaranteed that light can pass through the opening 20 and that a fluid-tight microscope chamber 26 is established.

The annular contact area 24 of the base plate 14 and thus the transparent disc 22 is surrounded by an annular projection 28 extending in vertical direction z of the holding device 12. The annular projection 28 is configured to act as a kind of tension ring for the membrane 2 if the holding device 12 is closed. Preferably, the tension ring 28 has an outer diameter that is equal or almost equal to an outer diameter of the membrane 2, or in other words the tension ring 28 has an inner diameter that is equal or almost equal to an outer diameter of the transparent disc 22. In order to create a smooth transition from the upper side 40 of the transparent disc 22 to a tension or guide surface 42 of the tension ring 28, a distance in vertical direction z between the contact area 24 and the tension surface 42 is equal or almost equal to the thickness of the transparent disc 22. By means of this, the membrane 2 rests step-free on the transparent disc 22 and on the tension ring 28, both in opened state and in closed state of the holding device 12.

The tension surface 42 decreases to an annular clamp surface 44 for receiving a bottom surface 45 of the frame 4. In vertical direction z , the clamp surface 44 is at a level that is lower than the level of the upper side 40 of the transparent disc 22. The distance in vertical direction z between these levels depends on various factors, such as the outer diameter of the membrane 2 and its thickness , thereby defining the resulting tension force Ft applied to the membrane 8 in the closed state of the holding device 12. In the shown embodiment , the clamp surface 44 is 0 , 5 mm below the upper side 40 of the transparent disc 22 in vertical direction z.

In order to receive the feet 6 of the frame 4, the clamp surface 44 is surrounded by a plurality of slots 46. The plurality of slots 46 enables the insert 1 to be placed in a very simple manner without a strict rotation angle regarding a vertical axis . The depth of the slots 46 is such that the feet 6 are in a distance to the slot grounds 48 in both states. By means of this, in the closed state it is guaranteed that the insert 1 is pressed against the clamp surface 44 of the base plate 14.

The plurality of slots 46 is surrounded by an annular wall 50 that limits the central portion of the base plate 14 as a kind of external border in radial direction.

Beyond the annular wall 50, a frame-like rectangular recess 52 is established for receiving a corresponding centre portion 54 of the cover 16. The centre portion 54 of the cover 16 will be explained in figures 10b and 12 in detail . The recess 52 has a flat ground 55 which is surrounded by a sidewall 56, The sidewall 56 ends at a lifted surface 58 which forms the bottom of the afore-mentioned recess 34 for placing the cover 16 in order to avoid a misalignment in longitudinal direction x or in transverse direction y. In order to facilitate the positioning of the cover 16 on the base plate 14, the sidewall 56 can be inclined in such way that the hole 32 of the cover 16 is self- centring with the opening 20 if the holding device 12 is closed.

In order to place the handle adequately if the cover 16 is positioned in the recess 34, on one of the transverse sides of the base plate 14 a downgraded upper portion 60 is established in which the handle 30 fits .

In order to allow an unrestricted access to the tissue section of the insert 1, on both longitudinal sides of the base plate 14 access grooves 62, 63 for instruments such as

microelectrodes are provided opposite to each other.

In figures 10a, 10b, 11 and 12, a more detailed explanation of the cover 16 follows. Figure 10a illustrates a top view, figures 10b and 11 illustrate a bottom view and figure 12 represents a sectional view along line BB in figure 11. It is made of a rigid material. Preferably, the cover 16 is produced via additive manufacturing such as laser sintering or 3D- printing . In order to avoid any drawback on the tissue section 3 , the base plate 14 is made of a biocompatible material, in particular a 3D-printable biocompatible polymeric material . A preferred 3D-printing method is stereolithography. The cover 16 has also a plate like shape with a large extension in

longitudinal direction x and a short extension in transverse direction y .

The rectangular centre portion 54 of the cover 16 providing the hole 32 is surrounded by an edge region 64. The edge region 64 is a material reduction in vertical direction z of the cover 16 and, in the shown embodiment , has the same thickness as the handle 30, The edge region 64 defines the outer rectangular form of the cover 16 and is of such an extent that it fits into the recess 34 of the base plate 14 preventing a shifting in longitudinal direction x or transverse direction y in both states. In order to support the self Centring of the cover 16 on the base plate 14, the centre portion 54 has two opposite angular faces 66, 68 in transverse direction y. The angular faces 66, 68 extend from a bottom surface 70 of the centre portion 54 to a flat underside 72 of the edge region 64. They are inclined correspondingly to the inclined sidewalls 56 of the base plate 14.

In order to have unrestricted instrument access to the tissue section 3 in the insert 1, the cover frame 64 has two opposite access grooves 74, 76 on its longitudinal sides which extent in transverse direction y . In the closed state of the holding device 12, the access grooves 74, 76 of the cover 16 are aligned with the access grooves 62, 63 of the base plate 14.

On the bottom surface 70 of the centre portion 54, a

cylindrical recess 78 is provided. The cylindrical recess 78 creates space for the annular wall 50 of the base plate 14 and thus for the insert 1 in the closed state of the holding device 12. For this purpose, the cylindrical recess 78 has an inner diameter that is slightly larger than an outer diameter of the annular wall 50 of the base plate 14.

On an upper surface 80 of the centre portion 54, the

cylindrical recess 78 is partially opened by the hole 32, The hole 32 has an ellipsoid shape and is limited by two opposite and horizontal projections 82, 84. At their opposite ends, the projections 82,84 are in contact. The orientation of the hole 32 is such that it has a longer extension in transverse direction y than in longitudinal direction x which benefits the unrestricted access for instruments to the tissue section 3 of the insert 1. On their lower sides, the projections 82, 84 form clamp surfaces 86, 88 for clamping the insert 1 between the base plate 14 and the cover 16 it the closed state of the holding device 12. In order to introduce the closing force Fc properly into the frame 4 of the insert 1, the clamp surfaces 86, 88 have a corresponding shape to an upper surface 89 of the frame 4 they are in contact with if the holding device 1 is closed.

In order to supply a fluid such as a buffer solution or nutrient solution to the tissue section 3 during examination, at least one inlet channel 90 and an outlet channel 92 are provided. They are positioned opposite to each other and extend in longitudinal direction x . They are integrated in the projections 82 , 84, wherein their inlet port 94 and outlet port 96 adapted to install a fluid connection to an external fluid system are positioned outside the projections 82 , 84 within the centre portion 54 of the cover 16 ,

As illustrated in figure 13, 14 and 15, the cover 16 comprises a heating element 98. The heating element 98 is provided for heating up the fluid, in particular a buffer or nutrient solution, supplied to the tissue section 3 during examination and to maintain a required temperature of the fluid.

The heating element 98 is positioned within the hole 32 and defines an examination area 100 of the tissue section 3. That means , during examination, the heating element 98 surrounds the tissue section 3 in the insert 1.

The examination area 100 has an ellipsoidal shape corresponding to the shape and orientation of the hole 32. Due to this , the examination area 100 has a larger extension in transversal direction y than in longitudinal direction. In transversal direction, it is spaced apart from opposite base plate

portions , such that two sickle-shaped gaps 97 , 99 are formed.

It has a ring-like hollow structure and is fully integrated in the cover 16. In order to incorporate the heating element 98 in a hot fluid water fluid system, a fluid inlet 102 and a fluid outlet 104 are provided on the upper surface 80 (see also figure 10a) . In the shown preferred embodiment, the hot fluid is hot water, so that for better understanding, the hot fluid system is also entitled as hot water system, and the fluid inlet 102 and the fluid outlet 104 are also entitled as water inlet 102 and fluid outlet 104. The water inlet 102 and the water outlet 104 are in fluid connection with the heating element 98 via two fluid channels 106, 108, which are entitled as water channels 106, 108 respectively. The water inlet 98 and the water outlet 104 are positioned lateral to the inlet port 94 for supplying the buffer and/or nutrient solution.

The heating element 98 has an angular internal peripheral surface 110. The internal peripheral surface 110 is suchlike that the examination area 100 is widened from the bottom to the top. Thus, the heating element seems to have a wedge-shaped internal peripheral surface 110. The peripheral surface 110 of the heating element 98 therefore mirrors the cone/wedge-shaped form of high numerical water immersion microscope objectives lenses with short working distances. During examination the objective lens is vertically ( Z) inserted and positioned above the examination area 100 in order to gain visual access to the tissue section 3.

External peripheral surfaces 112 , 114 of the heating element 98 are suchlike that they correspond to an inner circumferential frame shape of the used insert 1.

On its bottom, a flat ring surface 116 is provided. At a radial outer position of the ring surface 116, a ring projection 118 is provided. The ring projection 118 acts as a guiding element to smooth the guiding of the membrane 2 of the insert 1 in the closed state. In the closed state of the holding device 12, only the ring projection 118 of the heating element 98 is in contact with the membrane 2, the other outer surfaces of the heating element 98 such as the ring surface 118 and the external peripheral surfaces 112, 114 are in a distance to the frame 4 of the insert 1 and to the membrane 2, respectively.

In cross sectional view, preferably, the heating element 98 has a cavity 120 for receiving the hot water that corresponds to its outer shape. That means, in the shown embodiment, the cavity mainly has a trapezoidal shape. Due to the ellipsoidal shape of the hole 32 and the examination area 100, the cavity shape and volume is not constant over the circumference of the heating element 98 (see figure 12 and 15) .

As illustrated in figure 13, in order to control important biological , chemical or physical parameters of the tissue section 3 or the buffer fluid, such as temperature, oxygen saturation or pH value, not shown sensors can be provided. For this purpose, the cover 16 has respective engagement means, respectively engagement ports 103, 105. In the shown exemplary embodiment, the engagement means 103, 105 are four notches on the upper surface 80 of the cover 16. Preferably, the engaged sensors are in a maximum distance to their adjacent sensors. As shown, this can be reached by providing the notches 103, 105 diametrical opposite to each other.

To summarize, reference is made to figures 16 and 17. in the opened state of the holding device 12, the insert 1 which is positioned on the base plate 14 in the opening 20 rests solely on its membrane 2. In the preferred embodiment, the membrane 2 rests on the upper side 40 of the transparent disc 22 and on the tension surface 42 of the tension ring 28. Its frame 4 including its feet 6 are in a vertical distance to the base plate 14,

In order to close the holding device 12, the cover 16 is moved downwards on the frame 4. The cover 16 contacts an upper side 40 of the frame 4 via its clamp surfaces 86, 88 and presses the frame 4 with its bottom surface 45 against clamp surface 44 of the base plate 14. Due to the downward movement of the cover 16 and thus of the frame 4, the membrane 2 is drawn over the tension surface 42 of the tension ring 28. In addition, the ring projection 118 of the heating element 98 touches the membrane 2 and acts in corporation with the tension surface 42 as diverting means for the membrane 2,

In the closed state of the holding device 12, a closing force Fc is applied via the magnets and the frame 4 is clamped with its upper surface 89 and bottom surface 45 between the clamp surfaces 44, 86, 88 of base plate 14 and the cover 16. The feet 6 are received in the slots 46 and spaced apart from the slot grounds 48. As a consequence, a tension force Ft is applied to the membrane 2 which is tensioned accordingly . In the shown preferred embodiment , the tension force Ft is orthogonally orientated to the closing force Fc. While the closing force Fc acts in vertical direction z, the tension force Ft acts in horizontal direction, and even more radially on the membrane 2 and thus over its entire circumference . This prevents that shear forces are introduced into the membrane 2 due to the tensioning .

As also illustrated in figure 17 and as also mentioned above , in the closed state of the holding device 12, the heating element 98 is with its ring surface 118 and its external peripheral surfaces 112 , 114 in a distance to the frame 4 of the insert 1 and to the membrane 2 , respectively. Again, only the ring projection 118 of the heating element 98 touches the membrane 2 and acts in corporation with the tension surface 42 of the tension ring 28 as diverting means for the membrane 2,

To avoid any shifting of the cover 16 in longitudinal direction x or in transverse direction y, the clamp surfaces 44 , 86, 88 of the base plate 14 , and the bottom surface 44 of the frame 4 as well as the upper surface 89 of the frame 4 are flat and without any angle setting in their horizontal plane defined by the longitudinal direction x and transverse direction y. Due to the positioning of the ports 94, 96, 102, 103, 104, 105 on opposite transverse sides of the cover 16 and the port- free access between them in the direction of the large extent of the examination area 100, unrestricted access for required

instruments such as micropipettes is provided during

examination . Thus, the cover 16 has preferably two sections for ports 94, 96 (buffer solution), 102, 104 (hot fluid) and ports 103, 105 for additional measuring means such as a temperature sensor, an oxygen sensor or a pH sensor and two sections 74, 76 for access of scientific instruments during examination.

Disclosed are a holding device for holding a cell culture insert for microscopy applications, wherein the insert has a membrane which is configured to be provided with the biological sample and which is surrounded by a frame, wherein the insert can fully be received in the holding device and in a closed state of holding device the insert is fixed and the membrane is tensioned such that spontaneous displacements of the membrane are prevented, and a microscopy chamber with an integrated holding device.

Furthermore, the holding device provides a heating element with a shape adapted to the frame structure of the culture insert located in close proximity to the biological sample in a closed state of the holding device. Therefore, the heat transfer is optimized in a way that prevents undersaturation of to external buffers or nutrient solutions with oxygen and carbon dioxide.

Reference list

1 cell culture insert

2 membrane

3 biological sample / tissue section

4 sidewall / frame

6 foot

8 upper surface

10 lower surface

12 holding device

14 base plate

16 cover

18 retainment element

20 opening

22 transparent disc

24 contact area

26 microscope chamber

28 annular projection / tension ring

30 handle

32 hole

34 recess

36 recess

38 recess

40 upper side disc

42 tension surface

44 clamp surface

45 bottom surface frame

46 slot

48 slot ground

50 annular wall

52 frame-like recess

54 centre portion of the cover

55 ground of the centre portion

56 lifted surface

58 sidewall

60 downgraded upper portion

62 access grooves 63 access grooves

64 edge region

66 angular face

68 angular face

70 bottom surface

72 underside of the cover frame

74 access groove

76 access groove

78 cylindrical recess

80 upper surface

82 projection

84 projection

86 clamp surface

88 clamp surface

89 upper surface frame

90 buffer inlet channel

92 buffer outlet channel

94 buffer inlet port

96 buffer outlet port

97 gap

98 heating element

99 gap

100 examination area

102 fluid inlet / water inlet

103 engagement ports / engagement means / notches

104 fluid outlet / water outlet

105 engagement ports / engagement means / notches

106 fluid channel / water channel

108 fluid channel / water channel

110 internal peripheral surface

112 external peripheral surface

114 external peripheral surface

116 ring surface

118 ring projection

120 cavity

Fc closing force Ft tension force

X longitudinal direction

Y transverse direction

Z vertical direction