Preparation of samples for high pressure freezing

Abstract

The present invention relates to a device (10) for preparing a microscope sample (1) for a high-pressure freezing process, wherein the sample (1) is provided using an arrangement comprising an intermediate plate (33) of a high-pressure freezing box (30) and a culture chamber (100, 200), the intermediate plate (33) being attached to the culture chamber (100, 200) and being detachable from the culture chamber (100, 200) by achieving a relative movement between the intermediate plate (33) and the culture chamber (100, 200), and the sample (1) is provided On an encapsulating element (37) to be assembled into an opening (36) of an intermediate plate (33), wherein the device (10) comprises an engaging structure (11, 21), which is suitable for engaging with the intermediate plate (33) and limiting the movement of the intermediate plate (33) when the culture chamber (100, 200) is moved, so as to achieve the said relative movement between the intermediate plate (33) and the culture chamber (100, 200), thereby removing the intermediate plate (33) from the culture chamber (100, 200). In addition, the culture chamber (100, 200), the intermediate plate (33) of the high-pressure freezing box (30), the arrangement comprising these components and the corresponding method are also part of the present invention.

Description

Technical Field

[0001] The invention relates to a device for preparing microscopic samples for high-pressure freezing processes as well as means which can be used in conjunction with such a device and a corresponding method. Background Art

[0002] During optical microscopy imaging, living cells should be maintained under favorable environmental conditions. Stage incubators, stage heaters, CO2 chambers, etc. are common tools used for this purpose.

[0003] There are several stage incubators and chambers for conditioning the gas atmosphere around a sample during optical microscopy available. The operating principle of a particular type of incubation chamber is disclosed in DE 20 2016 007 488 U1, which provides the option of transferring samples between different locations while still maintaining environmental conditions.

[0004] In the following Figure 1 , components of a culture chamber of this type are shown in longitudinal section. The present invention is not limited to use with any particular type of culture chamber and provides Figure 1 For illustrative purposes only.

[0005] To assemble Figure 1 In the culture chamber 900 shown, an open silicone cylinder 110 is mounted to a glass plate forming the bottom 120 of the culture chamber. The cylinder 110 is closed by a glass cover 130, which may be additionally secured by a metal ring 140. The entire assembly is then covered by a metal cover 150, for example made of aluminum, containing a glass window 160. The gas to which the sample is exposed is introduced into the cover 150 via a feedthrough hole 170 or port connected to a gas supply means. From there, the gas maintains the atmosphere in the chamber by diffusion through the silicone cylinder 110. As shown in FIG. Figure 1 As shown, the culture chamber 900 can be used to culture a sample 1 , such as a layer of cells, in a liquid medium 2 .

[0006] It is usually desirable to capture the cell states observed in an optical microscope (especially in the culture chamber just mentioned) as quickly as possible (i.e., when the physiological event is detected) to preserve them for electron microscopy studies. In this regard, high-pressure freezing is a suitable tool for sample fixation, which will be explained in more detail below. However, for this method, the sample must be contained in a suitable sample holding means or carrier structure (commonly referred to as a "box") for the high-pressure freezing device. Transferring to these boxes is complicated and time-consuming, especially when the sample was previously contained in the culture chamber mentioned. This is the main obstacle to the rapid fixation of cell states in the methods and arrangements of the prior art.

[0007] The object of the present invention is to provide an advanced solution for high pressure freezing of samples kept under defined environmental conditions in an incubation chamber. Summary of the invention

[0008] Against this background, the invention proposes a device for preparing microscopic samples for high-pressure freezing processes, means that can be used in conjunction with such a device and a corresponding method according to the independent claims. Preferred embodiments are the subject matter of the dependent claims and the following description.

[0009] Here, if reference is made to a device "for preparing" a microscopic sample for a high-pressure freezing process, it is intended to refer to a device that is suitable for carrying out at least one step of a method for preparing a sample for a high-pressure freezing process as a whole. In this respect, the term "sample preparation" is intended to also include the incubation of the sample, in particular in a defined atmosphere. The device does not necessarily have to be used for the entire procedure, but can also be used for at least one step therein, since each step to some extent "prepares" the sample for a high-pressure freezing process. The same applies when referring to the method.

[0010] Before discussing the features and specific advantages of the present invention, some further details related to high pressure freezing and the problem of the method of transferring the sample from the incubation chamber to the high pressure freezer are presented in order to understand the basis of the present invention.

[0011] Water is the most abundant cell component and is therefore important for preserving the cell ultrastructure of biological samples. Currently, the only method for fixing cell components without introducing significant structural changes is cryogenic fixation. There are currently two commonly used methods: plunge freezing and high-pressure freezing. The present invention relates to high-pressure freezing.

[0012] In general, cryofixation has two distinct advantages over chemical fixation. It can be achieved within milliseconds and ensures simultaneous fixation of all macromolecular components. Many protein networks are very fragile and disintegrate at the slightest osmotic or temperature changes, and during cryofixation these unwanted effects are minimized. These techniques enable the study of biological samples with improved ultrastructural preservation and can facilitate the study of dynamic processes. Currently, the only way to vitrify thicker samples (up to 200 microns) is by high-pressure freezing.

[0013] Successful cryofixation (vitrification) should result in the transformation of water from a liquid to an amorphous solid state without inducing the nucleation of ice crystals. The nucleation of ice crystals depends on temperature and pressure. Crystallization also depends on the cooling rate, since freezing is a time-dependent process. The cooling rate depends on the thermal properties of water, the sample thickness and the heat extraction flow rate at the specimen surface. Therefore, high-pressure freezing is performed at high pressure and with a high flow rate of a refrigerant (especially liquid nitrogen).

[0014] For high pressure freezing, in other words, the biological sample is brought into contact under high pressure with a refrigerant provided at a cryogenic temperature.

[0015] Since the thickness of biological or other aqueous preparations for high pressure freezing should usually or generally not exceed 200 microns, the thickness of the sample must be limited. In addition, the sample must be protected from the flow of the cooling medium. For this purpose, a covering element consisting of a metal sample carrier or a sapphire plate or other suitable material is placed at a certain distance from the sample carrier carrying the sample, and the carrier carrying the sample can be, for example, a sapphire plate. At present, the carrier, the intermediate ring and the cover ring are manipulated with tweezers.

[0016] Devices for high-pressure freezing of biological and industrial samples are marketed by Leica Microsystems under the names "EM ICE", "EMHPM100" and "EM PACT". For example, A. Kaech and U. Ziegler, "High-pressure freezing: current status and future prospects", 2014, Volume 1117, DOI 10.1007 / 978-1-62703-776-1_8, pages 151 to 171, Chapter 8 of John Kuo (ed.), Electron Microscopy: Methods and Protocols, Methods in Molecular Biology, describes such devices.

[0017] With these devices, it is possible to cool a sample to cryogenic temperatures, in particular to temperatures below -100°C, with liquid nitrogen at a pressure of up to 2,100 (two thousand one hundred) bar, within a few milliseconds. In these devices, as mentioned above, a sample box is used to hold the sample during high-pressure freezing. The sample box can be made of, for example, high-strength plastic and can include three parts, namely two semi-cylinders with channels suitable for supplying a refrigerant flow from the high-pressure freezing device and a sample holding arrangement, which is usually called an "intermediate plate" with an opening for holding the sample, and the intermediate plate is sandwiched between the semi-cylinders. As mentioned above, the present invention is not limited to a specific type of sample box, as long as it is compatible with the solution proposed by the present invention.

[0018] In the intermediate plate, the sample itself is enclosed between two sufficiently thin and therefore thermally conductive material (such as sapphire, aluminum or copper) disks within the opening of the intermediate plate, wherein the disks themselves include recesses to receive the sample, or are separated by spacer rings to form a holder for the sample. The pressure at the sample position is generated by a refrigerant, which is pressurized to, for example, 2,100 bar for this purpose. More details in this regard will be explained with reference to the following figures. If an "intermediate plate" is mentioned below, this is intended to refer to a substantially flat element or arrangement of elements that is suitable for providing a space for receiving a sample to be vitrified. In particular, in the assembled state, the intermediate plate consists at least of a substantially flat structure having an opening in which a suitable disk can be arranged to form a sample space. These disks can be formed of various materials and do not need to be transparent. The term "intermediate plate" can be replaced by "carrier element" in any case used.

[0019] The incubation chambers for maintaining physiologically favorable environmental conditions described at the outset are designed for use with slides or cover slips as standard sample carriers. High-pressure freezing of samples contained in such incubation chambers therefore necessarily involves transferring the samples to an intermediate plate of a high-pressure freezing box. This is obviously associated with a disturbance of the environmental conditions in the incubation chamber to which the samples are exposed and may produce unwanted physiological reactions. Moreover, such a step is time-consuming and may therefore result in unwanted time lags, which make the observation of rapid physiological changes impossible.

[0020] One aspect of the invention therefore relates to an advantageous solution in which an intermediate plate of a high-pressure freezing box is mounted in an open state at the bottom of an incubation chamber of the type described above. The "open state" of the intermediate plate is to be understood as a state in which one of the above-mentioned trays (hereinafter referred to as "encapsulation element") is mounted in an opening of the intermediate plate, while another tray has not yet been mounted there. Thus, a sample placed on the already mounted tray can be removed from the incubation chamber and, after mounting another tray to close and seal the sample space, can be directly and quickly transferred together with the rest of the high-pressure freezing box to a high-pressure freezer.

[0021] Another aspect of the invention relates to the step of removing the intermediate plate mounted at the bottom of the culture chamber from the culture chamber. In an arrangement not designed according to the invention, such separation and transfer requires many manual steps, which are correspondingly time-consuming. The present invention solves this problem by simply moving the intermediate plate and the sample together from the culture chamber, wherein the intermediate plate is held fixed by a temporary holding structure. Once disassembled, the intermediate plate can be located in the immediate vicinity of the loading area of ​​the high pressure freezer, or even in its dedicated loading station.

[0022] Said closing of the intermediate plate or more specifically of its sample space, which comprises the careful fitting of another disk into the opening forming the sample space and optionally the fitting of a spacer ring and a gasket ring, requires skill and involves several maneuvering steps and is therefore very time-consuming in conventional methods. Therefore, an important aspect of the invention relates to a device comprising placing the intermediate plate in its open state in a geometrically well-defined position and placing another disk, preferably a single metal element, quickly and safely into the opening with the aid of a rotating arm.

[0023] Features and advantages of the present invention

[0024] According to the invention, a device for preparing microscopic samples for a high-pressure freezing process is proposed. The device provided according to the invention may be a high-pressure freezing device suitable for performing all or at least part of the steps of high-pressure freezing, and may therefore also include a so-called "loading station" suitable for assembling a high-pressure freezing box (e.g., as follows Figure 2 and 3 As shown in the figure), and the box is inserted into a space or chamber, wherein the high-pressure refrigerant passes through the box. Therefore, the device can also include means for providing high-pressure refrigerant and passing it through the chamber. Therefore, the device can include all or a subset of the means for high-pressure freezing processes according to the prior art or expert literature (such as Kaech and Ziegler (see above)). However, the device provided according to the invention can also be a dedicated device separate from the high-pressure freezing device, that is to say, as a separate structural unit, and can exactly contain the components described below, and optionally include other components. In the latter case. The dedicated device can, for example, be placed next to the high-pressure freezing device.

[0025] According to the invention, the device is used in combination with the cultivation of the sample in a cultivation chamber as already described at the outset or in a cultivation chamber of a similar type. According to the invention, as stated more generally previously in connection with the basic or important aspects of the invention, the sample is provided using an arrangement which comprises an intermediate plate of a high-pressure freezing box and such a cultivation chamber, wherein the intermediate plate is attached to the cultivation chamber. Thus, according to the invention, it is no longer necessary to laboriously transfer the sample to the intermediate plate of the high-pressure freezing box, which is necessary when using arrangements according to the prior art. This significantly reduces the disturbance of the environmental conditions to which the sample is exposed in the cultivation chamber and reduces or avoids unwanted physiological reactions. Since, according to the invention, the sample can already be provided on the corresponding intermediate plate, the time lag between cultivation and high-pressure freezing is also reduced, so that specific points of rapid physiological changes can also be observed.

[0026] In the context of the present invention, the intermediate plate is attached to the cultivation chamber so as to be removable from the cultivation chamber by achieving a relative movement between the intermediate plate and the cultivation chamber, as described in further detail below. This disassembly according to the invention achieved by a simple movement, wherein the intermediate plate is held fixed by a holding structure, hereinafter referred to as "engaging structure", reduces the time and effort of separating the cultivation chamber and the intermediate plate, so that the intermediate plate can be transferred to the high-pressure freezer particularly quickly. Once disassembled, the intermediate plate can be located in the immediate vicinity of the loading area of ​​the high-pressure freezer, or even in its dedicated loading station, or the disassembly can be achieved in a structure or equipment component located in the immediate vicinity of the high-pressure freezer or as a part of the high-pressure freezer.

[0027] As mentioned above, in the context of the present invention, the intermediate plate of the high-pressure freezing box is mounted in the open state on the bottom of the incubation chamber of the above-mentioned type. That is, the sample is provided on the encapsulation element that fits into the opening of the intermediate plate. Therefore, the preparation of the intermediate plate for assembling the high-pressure freezing box basically only requires the insertion of a further encapsulation element to seal the sample, which can be realized in the device according to a particularly preferred embodiment of the present invention, as described below.

[0028] As mentioned, the device provided according to the invention comprises an engagement structure, which is suitable for engaging with the intermediate plate and limiting the movement of the intermediate plate when the culture chamber is moved, so as to achieve the said relative movement between the intermediate plate and the culture chamber and thereby to remove the intermediate plate from the culture chamber. In particular, the engagement structure can be provided so that the culture chamber to which the intermediate plate is attached can be positioned by hand so that the entire intermediate plate or its structure is at least partially inserted into or in contact with the engagement structure. By a simple movement of the culture chamber, preferably also by hand, the intermediate plate can be removed from the culture chamber, remaining at least partially inserted into or in contact with the engagement structure.

[0029] According to a particularly preferred embodiment of the invention, which has already been briefly discussed above, the device provided according to the invention is suitable for holding an intermediate plate removed from a culture chamber in place in a processing position. Such a position can be defined in particular by a coupling structure or other positioning elements (such as stops, fences, recesses, frames, etc.) for removing the intermediate plate from the culture chamber. The device comprises a pivoting arm with a container, which is suitable for receiving another encapsulation element to be assembled in the opening of the intermediate plate so as to encapsulate the sample therein. In this embodiment, the pivoting arm serves as a means to accurately position the encapsulation element with which the sample is sealed in the opening of the intermediate plate, thereby eliminating or greatly reducing the technology traditionally required in this regard.

[0030] In the present invention, it is also possible to reduce the number of spacer rings and gasket rings to be fitted in the openings of the encapsulating elements, or to dispense with these elements altogether, thereby further reducing the time and effort required for sample preparation for cryofixation. This may be a result of the pivoting arm provided according to the invention, which may allow the use of suitable encapsulating elements, which may be press-fitted into the openings of the intermediate plate without gaskets, which is not possible by hand using tweezers. In order to reduce the need for spacers, at least one of the encapsulating elements may be provided with a recess forming the sample chamber, or in other words, with an outer ring protruding from the surface of the encapsulating element.

[0031] For the above-mentioned purposes and in order to provide the advantages of the present invention just discussed, in a preferred embodiment of the present invention just discussed, the pivot arm is pivotable between a loading position and an insertion position, in which another packaging element can be inserted into the container of the pivot arm, and in the insertion position, the other packaging element in the container of the pivot arm is placed in the opening of the intermediate plate held in place at the processing position.

[0032] In a first group of embodiments of the present invention, the middle plate of the high pressure freezer is attached to the lower surface of the bottom of the culture chamber by using at least one of mechanical and magnetic attachment means (such as a magnet, a bracket, a clamp, etc. inserted into the bottom of the culture chamber). In this first group of embodiments, the bottom of the culture chamber is provided with an opening that provides access to the opening of the middle plate, but is not itself used for attaching the middle plate. In this embodiment, the upper surface of the middle plate is directly attached to the lower surface of the culture chamber. A sealing element such as a sealing ring may be provided between the two surfaces to prevent leakage of the culture fluid from the culture chamber.

[0033] In a first group of embodiments, the intermediate plate is detachable from the cultivation chamber by achieving said movement in the form of at least one of the following movements: linear and / or rotational movement of the cultivation chamber in a plane corresponding to the lower surface of the bottom of the cultivation chamber; tilting movement of the cultivation chamber about an axis parallel to the lower surface of the bottom of the cultivation chamber; and lifting movement of the cultivation chamber in a direction at an angle to the lower surface of the bottom of the cultivation chamber. The engagement structure is particularly suitable for engaging with the intermediate plate to restrict said at least one of the linear, rotational, tilting and lifting movements.

[0034] In order to limit the linear movement, a simple stopper or fence may be provided as an engagement structure, so that when the intermediate plate is attached to the bottom surface of the cultivation chamber and protrudes therefrom, the intermediate plate may be prevented from moving when the cultivation chamber is pushed onto the stopper or fence and stays at a position limited by the stopper or fence. In such a configuration, the means for attaching the intermediate plate to the cultivation chamber are designed to provide an attachment force that can be overcome by a corresponding pushing force, for example achieved by hand.

[0035] In order to limit the rotational movement, a recess or at least two fences may be provided as an engagement structure into which or between which the intermediate plate attached to the bottom surface of the cultivation chamber and protruding therefrom fits. The cultivation chamber is rotated, whereby the intermediate plate attached to the bottom surface of the cultivation chamber is detached from the cultivation chamber. Also in this respect, the means for attaching the intermediate plate to the cultivation chamber are designed to provide an attachment force that can be overcome by a corresponding rotational force, for example achieved by hand.

[0036] In this case, one or more (especially two) protrusions (especially in the form of tenons or pins) can be provided as an engagement structure, as an additional engagement structure or as an alignment structure in addition to the engagement structure. One or more protrusions can be provided at the intermediate plate and at least one matching recess can be provided at its corresponding part, with which the culture chamber contacts to remove the intermediate plate, or one or more protrusions can be provided at the corresponding part and at least one matching recess can be provided at the intermediate plate.

[0037] In order to limit the tilting movement, the engagement structure can be configured as a structure with a lower cutout, into which a corresponding protrusion provided at the intermediate plate can be inserted, so that when the culture chamber is tilted while maintaining the protrusion of the intermediate plate inserted in the lower cutout, the overhang of the lower cutout "pulls" the intermediate plate from the culture chamber in a manner similar to a cap opener or bottle opener. The protrusion of the intermediate plate can be maintained inserted in the lower cutout by manually pressing the culture chamber with the intermediate plate into the direction of the lower cutout, or by providing a stopper that limits the removal from the lower cutout. In addition, here, in this regard, the means for attaching the intermediate plate to the culture chamber is designed to provide an attachment force that can be overcome by a corresponding tilting force, for example, achieved by hand.

[0038] The lifting movement can be limited by providing a set of parallel guide elements with undercuts (e.g. in the form of dovetail guides) which are adapted to slidingly receive complementary structures provided at the intermediate plate in the insertion direction. When the culture chamber is lifted in a direction at an angle to the insertion direction, and when a suitable attachment force is also selected here, the intermediate plate can thus be detached from the culture chamber.

[0039] It should be understood that in the present invention, in the first group of embodiments, the movement or movement trajectory is not necessarily limited to any one of linear, rotational, tilting and lifting movements, but for example, a rotational movement or a linear movement may be followed by a lifting movement, and so on.

[0040] Generally speaking, in the first group of embodiments, the intermediate plate may protrude from the bottom of the cultivation chamber when attached to the bottom of the cultivation chamber. The engagement structure may be provided as a recess having a shape complementary to at least a portion of the shape of the intermediate plate and adapted to receive at least a portion of the intermediate plate protruding from the bottom of the cultivation chamber. In this regard, the term "recess" should be understood in a broad sense and may be a recess on a flat surface, a recess between fence structures or a recess in front of a stopper, an undercut of a guide rail or a different structure, etc.

[0041] In a second group of embodiments of the present invention, the bottom of the culture chamber includes an opening with a guide structure, which is suitable for slidably receiving the intermediate plate. That is, compared with the first group of embodiments just discussed, the opening not only provides access to the opening in the intermediate plate, but also serves as an attachment structure. The guide structure suitable for slidably receiving the intermediate plate can be designed in particular as a set of dovetail guides, which are designed to cooperate with corresponding structures in the intermediate plate. In this second group of embodiments, the intermediate plate is detachable from the culture chamber by realizing the movement in the form of a linear movement defined by the guide structure, and the engagement structure includes a stopper to keep the intermediate plate in place when realizing the movement. In other words, the intermediate plate can be pushed out of the guide structure such as the dovetail guide by such movement. In this case, too, additional protrusions and recesses as described above can be provided.

[0042] In embodiments of the invention in which a pivoting arm adapted to place the enclosing element in place is provided, the container may be adapted to adhesively retain the further enclosing element to prevent displacement or falling out during the pivoting movement of the pivoting arm. The adhesive force is selected so that it can be overcome by the retaining force. With the retaining force, the further enclosing element is retained in the opening of the intermediate plate after insertion, for example in a pressed-fit form.

[0043] The invention also relates to a culture chamber suitable for use in an arrangement comprising a culture chamber and an intermediate plate of a high pressure freezer, the culture chamber comprising means for attaching the intermediate plate to the culture chamber such that the intermediate plate is detachable from the culture chamber by achieving a relative movement between the intermediate plate and the culture chamber. With regard to such a culture chamber and its specific features and advantages, reference is made to the above explanations. Each and any of the features described above may form part of a culture chamber provided according to the invention.

[0044] An intermediate plate (i.e. a sample holding means for high pressure freezing) suitable for use in a high pressure freezer box in an arrangement comprising a culture chamber and an intermediate plate is also part of the invention. The intermediate plate provided according to the invention comprises means for attaching the intermediate plate to the culture chamber such that the intermediate plate is detachable from the culture chamber by enabling a relative movement between the intermediate plate and the culture chamber. In addition, reference is made to the explanations above for the intermediate plate and its specific features and advantages. Each and any of the features described above may be part of an intermediate plate provided according to the invention.

[0045] According to a particularly preferred embodiment of the invention, the cultivation chamber can be manufactured together with the intermediate plate in a suitable process, in particular pressure forming or additive manufacturing (3D printing), wherein the breakable connection between the cultivation chamber and the intermediate plate is provided in the form of thin, weak or brittle structures forming intentional breaking points. By destroying these structures, the intermediate plate can be removed from the cultivation chamber essentially as described above, i.e. using the joining structures.

[0046] The above explanations also apply to the arrangement comprising a cultivation chamber and an intermediate plate of the high pressure freezing box provided according to the present invention.

[0047] The method provided according to the present invention is provided for preparing a microscope sample for a high pressure freezing process, wherein the sample is provided using an arrangement comprising an intermediate plate of a high pressure freezing box and a culture chamber, the intermediate plate being attached to the culture chamber and being detachable from the culture chamber by achieving relative movement between the intermediate plate and the culture chamber, and the sample is provided on an encapsulating element fitted into an opening of the intermediate plate. The method provided according to the present invention comprises using a device comprising an engagement structure, the engagement structure being adapted to engage with the intermediate plate and restrict movement of the intermediate plate when the culture chamber is moved, and the method comprises achieving engagement of the engagement structure with the intermediate plate and achieving said relative movement between the intermediate plate and the culture chamber, thereby detaching the intermediate plate from the culture chamber.

[0048] In addition, please refer to the above explanations for the method according to the present invention. The method according to the present invention utilizes the features and advantages of the apparatus and other components and arrangements described in the above preferred embodiments.

[0049] In a particularly preferred embodiment of the method provided according to the invention, the device is adapted to hold an intermediate plate removed from the culture chamber in place at the processing position, and the device comprises a pivoting arm with a container adapted to receive a further encapsulating element to be fitted in an opening of the intermediate plate for encapsulating the sample, wherein the pivoting arm is pivotable between a loading position, in which the further encapsulating element can be inserted into the container of the pivoting arm, and an insertion position, in which the further encapsulating element inserted into the container of the pivoting arm can be placed in the opening of the intermediate plate held in place at the processing position. The method comprises holding an intermediate plate removed from the culture chamber in place at the processing position and inserting a further encapsulating element using the pivoting arm of the device.

[0050] In any case, in the methods provided according to the present invention, the devices, culture chambers, intermediate plates and / or arrangements described in the different embodiments above can be used.

[0051] In the method according to the invention, providing the sample may comprise exposing the living matter to defined environmental conditions provided in a culture chamber, as is generally known for such culture chambers.

[0052] Other features of the invention will be described in conjunction with the accompanying drawings, in which embodiments of the invention are described in contrast to the prior art. It should be noted that the specific features of the embodiments described with respect to the accompanying drawings and described above may be used in any combination and / or alone without departing from the scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 An incubation chamber for culturing samples, which does not form part of the present invention, is shown in a simplified cross-sectional view.

[0054] Figure 2 A high pressure freezer box that may be used with the present invention is shown in a simplified exploded view.

[0055] Figure 3 An intermediate plate of a high pressure freezer box that can be used with the present invention is shown in a simplified cross-sectional view.

[0056] Figure 4 A culture chamber for culturing a sample according to an embodiment of the present invention is shown in a simplified cross-sectional view.

[0057] Figure 5 A culture chamber for culturing a sample according to an embodiment of the present invention is shown in a simplified perspective view.

[0058] Fig. 6A and Figure 6B The step of removing the intermediate plate from the cultivation chamber by tilting according to an embodiment of the present invention is shown.

[0059] Fig. 7A and Figure 7B The step of removing the intermediate plate from the cultivation chamber by lifting is shown according to an embodiment of the present invention.

[0060] FIG. 8A to FIG. 8E The steps of removing the middle plate from the cultivation chamber by sliding and sealing the opening according to an embodiment of the present invention are shown.

[0061] In the drawings, similar elements are marked with the same reference numerals, and repeated description thereof is omitted only for the sake of brevity. DETAILED DESCRIPTION

[0062] Figure 1 A culture chamber 900 for culturing samples, which does not form part of the present invention, is shown in simplified cross-sectional view and has been described at the outset. Please refer to the above description. It should also be noted that the present invention is not intended to be limited by the specific type of culture chamber shown here.

[0063] Figure 2A high pressure freezing box 30, i.e. a sample holding means, which can be used with the present invention is shown in an exploded view. The box 30 can be used in the high pressure freezing devices known in the art mentioned above. For example, reference is made to the literature on the "EM ICE", "EM HPM100" and "EM PACT" systems of Leica Microsystems cited above (e.g. reference is made to DE 10 2013003 164 A1, which further describes the details of the high pressure freezing systems) and scientific review literature, such as Kaech & Ziegler (see above). In the assembled state, the box 30 can be transferred to the high pressure freezing device by using a known holder. The handling of boxes such as the box 30 is widely described elsewhere and is not explained here for the sake of brevity.

[0064] The box 30 includes two holding elements 31, 32, each of which is substantially semi-cylindrical in shape. An intermediate plate 33 is arranged between the holding elements 31, 32 of the box 30. A refrigerant channel 34 is provided between the holding elements 31, 32, and the refrigerant channel 34 is formed by a groove in the longitudinal direction on the inner flat surface of the holding elements 31, 32. Via the refrigerant channel 34, a low-temperature refrigerant can be substantially along the longitudinal axis of the box 30 and Figure 2 The sample is conveyed in the direction of the dashed arrow 35 to be received or contained in the opening 36 forming the sample space in the middle plate 33 .

[0065] Figure 3 The longitudinal section along the vertical plane shows Figure 2 The intermediate plate 33 used in the box 30 shown is thus provided with an opening 36 (as in accordance with Figure 1 The sample space formed in the middle plate 33 is cut. The opening 36 is circular and arranged at the center or near the center of the middle plate 33, and the sample 1 is held between two encapsulation disks, which include a first (bottom) disk 37 and a second (top) disk 38, for example, circular encapsulation elements 37, 38 in the form of disks made of sapphire, metal or other suitable materials. The encapsulation elements 37, 38 are fixed in the opening 36, and their distance is adjusted by fixing means, sealing means and / or spacing means 39. The encapsulation elements 37, 38 themselves may also include one or more recesses, and when the encapsulation elements are inserted into the opening 36, the sample 1 can be received in the recess, thereby reducing or eliminating the need for fixing means, sealing means and / or spacing means 39. Since the rapid flow of the refrigerant required for cooling will carry away the sample, the encapsulation elements 37, 38 are also used to protect the sample 1.

[0066] In the prior art mentioned, further details and variations of the box 30 and its intermediate plate 33 are described. Likewise, it should be understood that the present invention is not limited to the specific configuration of the box 30 and the intermediate plate 33. The dimensions of the entire box 30 are designed so that the high pressure required for high-pressure freezing (e.g., more than 2,000 bar) can be established and maintained within a period of preferably 200 to 500 milliseconds, thereby achieving rapid freezing of the sample 1 within this time interval.

[0067] Figure 4 A culture chamber for culturing a sample according to an embodiment of the present invention (or the "first group of embodiments" as explained in more detail above) is shown in a simplified cross-sectional view.

[0068] Figure 4 The components of the culture chamber 100 shown are Figure 1 The culture chamber 900 shown is designed slightly differently, but may also be designed in the same manner. The present invention is not characterized by or limited by the presence or absence of specific parts of the culture chamber 900. Figure 4 The components of the culture chamber 100 shown are used with Figure 1 The culture chamber 900 shown is labeled with the same reference numerals.

[0069] Here, too, in order to assemble Figure 4 In the illustrated culture chamber 100, an open silicone cylinder 110 is mounted to a glass plate forming the bottom 120 of the culture chamber. The cylinder 110 is also closed by a glass cover 130. In the culture chamber 100, the entire assembly is covered by a metal cover 150 to which the glass cover 130 is fixed, and no separate glass window 160 is provided. The gas to which the sample 1 is exposed can be provided and the gas atmosphere can be maintained in any conceivable manner.

[0070] The middle plate 33 (for example, as previously combined Figure 2 and Figure 3 The intermediate plate 33 is attached to the lower surface of the bottom 120 of the culture chamber 100 by using any conceivable means (e.g., mechanical and magnetic attachment means that are not specifically shown for reasons of generality), and the intermediate plate 33 is detachable from the culture chamber 100 by enabling the movement of the culture chamber 100, as described in detail above for different embodiments. As shown, the intermediate plate 33 is provided in an open state, i.e., only the lower encapsulation element 37 is inserted, and the sample 1 is provided on the lower encapsulation element. The bottom 120 of the culture chamber 100 includes an opening 180 for providing a fluid passage to the opening 36 of the intermediate plate 33.

[0071] Figure 5 1 is a simplified perspective view showing a culture chamber for culturing samples according to another embodiment of the present invention (or the "second group of embodiments" as explained in more detail above). Figure 5The components of the culture chamber 200 shown may also be used with Figure 1 The culture chamber 900 or Figure 4 The cultivation chamber 100 shown is designed slightly differently, but can also be designed in the same way. Figure 5 The components of the culture chamber 200 shown are used with Figure 1 and Figure 4 The culture chamber 900 is shown with the same reference numerals as 100 .

[0072] exist Figure 5 In the illustrated cultivation chamber 200, the bottom 120 of the cultivation chamber 200 comprises an elongated opening 160 having a guide structure 165 suitable for slidably receiving the intermediate plate 33. As previously described, this may be a dovetail structure or any other guide structure suitable for holding the intermediate plate 33. In the cultivation chamber 200, the intermediate plate 33 is detachable from the cultivation chamber 200 by achieving a movement in the form of a linear movement defined by said guide structure 165, i.e. in particular parallel to the longitudinal extension of the guide structure 165, and the engagement structure (as described in further detail below) comprises, for example, a guide structure 165 which is described later on. Figure 8B , 8D and a stopper shown at 21 in FIG8E to hold the intermediate plate 33 in place when the movement is achieved. Similarly, the bottom 120 of the culture chamber 200 includes an opening for providing a liquid passage to the opening 36 of the intermediate plate 33, but the liquid passage is Figure 5 It is not visible in the figure because it is covered by the intermediate plate 33.

[0073] Fig. 6A and Figure 6B The step of removing the intermediate plate from the culture chamber by tilting according to an embodiment of the present invention is shown. The culture chamber is basically as Figure 4 The illustrated embodiment (or “first set of embodiments”) is implemented as described and is therefore labeled 100 .

[0074] Generally, since the middle plate 33 protrudes from the bottom 120 of the culture chamber 100 when attached to the bottom 120 of the culture chamber 100, the engagement structure 11 may be provided as a recessed portion having a shape complementary to at least a portion of the shape of the middle plate, the recess being adapted to receive at least a portion of the middle plate 33 protruding from the bottom 120 of the culture chamber 100. In the illustrated embodiment, in order to limit the tilting movement, the engagement structure 11 is provided as a structure having a lower cutout into which the corresponding protrusion 13 provided at the middle plate 33 may be inserted, so that when the culture chamber 100 is tilted (e.g., while the protrusion 13 of the middle plate 33 is maintained inserted into the lower cutout), the engagement structure 11 may be provided as a structure having a lower cutout into which the corresponding protrusion 13 provided at the middle plate 33 may be inserted, Fig. 6A When the middle plate 33 is pulled off by the overhanging portion of the lower cutout (as shown by the white arrow in FIG. 1 ), the middle plate 33 is “pulled off” by the overhanging portion of the lower cutout.

[0075] Fig. 7A and 7B 1 shows the steps of removing the intermediate plate from the culture chamber by lifting according to an embodiment of the present invention. Here, too, the culture chamber is basically as Figure 4 The illustrated embodiment (or “first set of embodiments”) is implemented as described and is therefore labeled 100 .

[0076] Here, too, in general, since the intermediate plate 33 protrudes from the bottom 120 of the culture chamber 100 when attached to the bottom 120 of the culture chamber 100, the joining structure 11 can be provided as a recess whose shape is complementary to at least a portion of the shape of the intermediate plate, and the recess is suitable for receiving at least a portion of the intermediate plate 33 protruding from the bottom 120 of the culture chamber 100.

[0077] The lifting movement can be specifically limited by providing a set of parallel undercut guide elements as engagement structures 11, for example in the form of dovetail guides, which are adapted to slidingly receive complementary structures provided at the intermediate plate in the form of protrusions 13 in the insertion direction. Fig. 7A and 7B In the illustration of FIG. , the insertion direction is orthogonal to the plane of the paper. When the culture chamber 100 is lifted in a direction that is angled with the insertion direction, as in accordance with Fig. 7A As indicated by the white arrow, the middle plate 33 can be removed from the cultivation chamber 100 .

[0078] exist 6A to 7B In the following figures, the device according to the present invention is shown in a partial view and is designated as 10. As mentioned previously, the device 10 can also be a high-pressure freezing device, suitable for performing all or at least part of the additional steps of high-pressure freezing, or the device 10 can be a dedicated device separate from the high-pressure freezing device, i.e. a separate structural unit. 6A to 7B The device 10 of 1 comprises an engagement structure 11, whereas the device 10 according to the following figure comprises an engagement structure 21. A surface of the device 10 is referenced 12.

[0079] FIG. 8A to FIG. 8E The steps of removing the middle plate from the cultivation chamber by sliding and sealing the opening in the middle plate according to an embodiment of the present invention are shown.

[0080] and Figures 6A to 7B In contrast, the culture room is basically Figure 5 The illustrated embodiment (or "second group of embodiments") is implemented as described and is therefore also referenced herein as 200.

[0081] Since in the cultivation chamber 200, the bottom 120 comprises an opening 160 having a guide structure 165 adapted to slidably receive the intermediate plate 33, and since the intermediate plate 33 is detachable from the cultivation chamber 200 by achieving a movement in the form of a linear movement defined by said guide structure 165, the engagement structure 21 may provide a stop to hold the intermediate plate 33 in place when said movement is achieved. In order to guide the cultivation chamber 200 in such a movement, a track structure 12 may be provided in the manner shown or of a similar design. Fig. 8A and 8B The bottom surface of the culture chamber 200 and the device 10 is shown ( Fig. 8A ) and the top surface of the culture chamber 200 and the device 10 ( Figure 8B ). As indicated by the arrows, the culture chamber 200 with the intermediate plate 33 attached can be placed on the device 10. In addition, as Fig. 8A and 8B As shown, matching protrusions and recesses 33a, 33b may be provided to improve proper alignment of the intermediate plate 33 and the device 10.

[0082] As also indicated by the arrows, Figure 8C The movement performed in order to remove the intermediate plate 33 from the cultivation chamber 200 is shown.

[0083] like Fig.8D As shown, the device 10 is suitable for holding the intermediate plate 33 removed from the culture chamber 200 in place at a processing position (as defined by the engagement structure 21), and the device 10 comprises a pivoting arm 24 having a container 26, which is suitable for receiving another (top) encapsulating element 38 to be fitted into the opening 36 of the intermediate plate 33, so as to encapsulate the sample 1 therein. Figure 8C As shown by the arrow in , the pivot arm 24 is pivotable between a loading position, in which a further encapsulating element 38 can be inserted into the receptacle 26 of the pivot arm 24, and an insertion position, in which a further encapsulating element 38 in the receptacle 26 of the pivot arm 24 is placed in an opening 36 of the intermediate plate 33 held in place in the processing position. For pivoting the pivot arm 24, a knob 25 may be provided. Fig. 8E It is shown (likewise indicated by arrows) how the intermediate plate 33 can be removed from the device 10 after a further enclosing element 38 has been placed in the opening 36 and the pivot arm 24 has been swung back into the loading position.

Claims

1. A device (10) for preparing a microscope sample (1) for a high-pressure freezing process, wherein the sample (1) is provided using an arrangement comprising an intermediate plate (33) of a high-pressure freezing box (30) and a culture chamber (100, 200), the intermediate plate (33) being attached to the culture chamber (100, 200) and being detachable from the culture chamber (100, 200) by achieving a relative movement between the intermediate plate (33) and the culture chamber (100, 200), and the sample (1) is provided on an encapsulation element (37) fitted into an opening (36) of the intermediate plate (33), wherein The device (10) comprises a coupling structure (11, 21), wherein the coupling structure (11, 21) is adapted to couple with the intermediate plate (33) and restrict movement of the intermediate plate (33) when the culture chamber (100, 200) is moved, so as to achieve the relative movement between the intermediate plate (33) and the culture chamber (100, 200), thereby removing the intermediate plate (33) from the culture chamber (100, 200).

2. The device (10) according to claim 1, wherein the device (10) is suitable for holding the intermediate plate (33) removed from the culture chamber (100, 200) in place at a processing position, and wherein the device (10) comprises a pivoting arm (24) having a container (26) suitable for receiving a further encapsulation element (38) to be fitted into the opening (36) of the intermediate plate (33), so as to enclose the sample (1) therein, The pivot arm (24) is pivotable between a loading position and an insertion position, wherein the further encapsulating element (38) is insertable into the container (26) of the pivot arm (24), and wherein the further encapsulating element (38) in the container (26) of the pivot arm (24) is placed in the opening (36) of the intermediate plate (33) held in place at the processing position.

3. The device (10) according to claim 1 or 2, wherein the intermediate plate (33) is attached to the lower surface of the bottom (120) of the culture chamber (100) by using at least one of mechanical and magnetic attachment means, wherein the intermediate plate (33) is detachable from the culture chamber (100) by realizing the movement in the form of at least one of the following movements: a linear movement defined by a guide structure (165) and / or a rotational movement of the culture chamber (100); a tilting movement of the culture chamber (100) around an axis parallel to the lower surface of the bottom (120) of the culture chamber (100); and a lifting movement of the culture chamber (100) in a direction at an angle to the lower surface of the bottom (120) of the culture chamber (100), The engagement structure (11, 21) is adapted to engage with the intermediate plate (33) to restrict at least one of linear, rotational, tilting and lifting movements.

4. The device (10) according to claim 3, wherein the intermediate plate (33) protrudes from the bottom (120) of the cultivation chamber (100) when attached to the bottom (120) of the cultivation chamber (100), and wherein the engagement structure (11, 21) is provided as a recess, the shape of which is complementary to at least a part of the shape of the intermediate plate and is suitable for receiving at least a part of the intermediate plate (33) protruding from the bottom (120) of the cultivation chamber (100).

5. The device (10) according to claim 1 or 2, wherein the bottom (120) of the cultivation chamber (200) comprises an opening having a guide structure (165), the guide structure (165) being suitable for slidingly receiving the intermediate plate (33), wherein the intermediate plate (33) is detachable from the cultivation chamber (200) by achieving the movement in the form of a linear movement defined by the guide structure (165), wherein the engagement structure (11, 21) comprises a stop to keep the intermediate plate (33) in place when achieving the movement.

6. The device (10) of claim 2, wherein the container (26) is adapted to adhesively retain the further enclosing element (38).

7. A cultivation chamber (100, 200) adapted for use in an arrangement comprising the cultivation chamber (100, 200) and an intermediate plate (33) of a high-pressure freezer (30), the cultivation chamber (100, 200) comprising means for attaching the intermediate plate (33) to the cultivation chamber (100, 200) such that the intermediate plate (33) is detachable from the cultivation chamber (100, 200) by achieving relative movement between the intermediate plate (33) and the cultivation chamber (100, 200).

8. An intermediate plate (33) of a high-pressure freezer box (30), suitable for use in an arrangement comprising a culture chamber (100, 200) and the intermediate plate (33), the intermediate plate (33) comprising means for attaching the intermediate plate (33) to the culture chamber (100, 200), so that the intermediate plate (33) is detachable from the culture chamber (100, 200) by achieving relative movement between the intermediate plate (33) and the culture chamber (100, 200).

9. An arrangement comprising a cultivation chamber (100, 200) according to claim 7 and an intermediate plate (33) of a high-pressure freezer box (30) according to claim 8.

10. A method for preparing a microscope sample (1) for a high-pressure freezing process, wherein the sample (1) is provided using an arrangement comprising an intermediate plate (33) of a high-pressure freezing box (30) and a culture chamber (100, 200), the intermediate plate (33) being attached to the culture chamber (100, 200) and being detachable from the culture chamber (100, 200) by achieving a relative movement between the intermediate plate (33) and the culture chamber (100, 200), and the sample (1) is provided on an encapsulation element (37) fitted into an opening (36) of the intermediate plate (33), wherein The method comprises using a device (10) comprising a coupling structure (11, 21), wherein the coupling structure (11, 21) is adapted to couple with the intermediate plate (33) and restrict movement of the intermediate plate (33) when the culture chamber (100, 200) is moved, and wherein the method comprises achieving coupling of the coupling structure (11, 21) with the intermediate plate (33), and achieving the relative movement between the intermediate plate (33) and the culture chamber (100, 200), thereby detaching the intermediate plate (33) from the culture chamber (100, 200).

11. A method according to claim 10, wherein the device (10) is suitable for holding the intermediate plate (33) removed from the culture chamber (100, 200) in place at a processing position, and wherein the device (10) comprises a pivot arm (24), the pivot arm (24) having a container (26), the container (26) being suitable for receiving a further encapsulation element (38) to be fitted into the opening (36) of the intermediate plate (33), thereby encapsulating the sample (1), wherein the pivot arm (24) is pivotable between a loading position and an insertion position, in which, The further encapsulating element (38) can be inserted into the container (26) of the pivot arm (24), and in the inserted position, the further encapsulating element (38) inserted into the container (26) of the pivot arm (24) can be placed in the opening (36) of the intermediate plate (33) maintained in place at the processing position, and wherein the method comprises maintaining the intermediate plate (33) removed from the culture chamber (100, 200) in place at the processing position and inserting the further encapsulating element (38) using the pivot arm (24) of the device (10).

12. The method according to any one of claims 10 to 11, wherein providing the sample (1) comprises exposing living matter to defined environmental conditions provided in the culture chamber (100, 200).

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