Patent Application
20230160852
The invention relates to an electrophoresis device for use in a method for producing transparent biological samples.
Transparent biological samples (hereinafter also referred to as “preparations”, “tissue” or the like) are required in pathology and histology to enable the prepared tissue to be imaged, for example in three dimensions, for instance by means of light sheet microscopy. In order to achieve sufficient transparency of the preparations for this purpose, those components that have a high absorption or that have a refractive index that differs from the refractive index of the tissue to be examined must be removed from the preparation. These components primarily include the heme groups of the blood pigment hemoglobin, as well as lipids from the biological tissues. The process of removing non-transparent substances and components from a tissue is referred to and known as clarifying or clarification (also known as “clearing”). Some of the methods of electrophoresis are used for this purpose; this procedure is then also referred to as “electroclearing”.
In conventional electrophoresis, the components (analytes) to be examined are separated according to their size or charge within the solid phase of a suitable carrier material and thus detected, the conductivity of the electrophoresis buffer being substantially determined by the ions previously dissolved in the buffer. The buffer (also referred to as the “reaction liquid”) has a high ionic strength. A contamination of the buffer by the analytes and a consequent change in the electric field does not usually occur in this case.
In contrast, known from DE 10 2016 123 458 B3 is a preparative electrophoresis method for clarification of tissue preparations in which the electrophoresis buffer has a low ionic strength. Here, the solid phase is the tissue from which the “contaminating” components are to be removed under the action of an electric field. In this case, positively charged ions migrate to the cathode, negatively charged ions to the anode. During their release from the tissue, the ions substantially determine the conductivity of the electrophoretic buffer. The clearing process can be followed quantitatively on the basis of the change in the conductivity of the buffer during electrophoresis. In order to allow the largest possible proportion of the electrophoretic force to act on the interfering components, or components to be removed, the ion concentration of the buffer must be appropriately low and kept constant. A low ion concentration also minimizes the flow of current and thus the heat generated, thereby making it possible to avoid thermal damage to the tissue.
Also known from the DE 10 2016 123 458 B3 is an electrophoresis chamber having a waisted reaction chamber that is rotationally symmetrical about a vertical axis and that can be filled with electrophoresis solution, and having a downwardly open annular channel into the reaction chamber, a first annular electrode in the reaction chamber and a second annular electrode in the reaction chamber above the waist. For the corresponding electrophoresis method, the ion concentration of the buffer must be appropriately low and kept constant. During the clarifying process, therefore, the contaminated electrode buffer must be exchanged for a new, non-contaminated buffer. The buffer is exchanged in this case by tipping the old buffer out of the reaction chamber, which is open at the top, and then admitting fresh buffer. This can damage the tissue, which is often very sensitive to mechanical effects, for which reason it should be removed from the reaction frame before every buffer change. However, unwanted impairment of the tissue quality can also occur during the step of removing the sample. In addition, this additional work step is time-consuming.
The invention is therefore based on the object of providing an electrophoresis device, for use in a method for producing transparent biological samples, that eliminates the disadvantages in the prior art and that above all enables the reaction liquid to be changed quickly and easily. The electrophoresis device provided is intended in this case, in particular, to ensure a changing of the reaction liquid that is as gentle as possible for the biological sample.
To achieve the object, the invention provides an electrophoresis device for use in a method for producing transparent biological samples, comprising a reaction frame, wherein the reaction frame has an open top side and a bottom side opposite the open top side. The device is characterized in that the bottom side at least partially comprises an opening. A reaction frame is preferably provided, the bottom side of which is designed entirely as an opening. Such a reaction frame therefore has no base part connected to the reaction frame. The top side of the reaction frame in this case is to be understood as the side that is arranged above a horizontal central axis of the reaction frame. Accordingly, the bottom side is arranged below the horizontal central axis of the reaction frame.
An electrophoresis device designed in this way is advantageous for the clearing method because the reaction liquid required for the method can be exchanged quickly and easily. For this purpose, it may be provided, for example, that the reaction frame is received in a receiving vessel filled with reaction liquid, or buffer, for which purpose the receiving vessel must have a base plate. The receiving vessel in this case may be, for example, a tank or a hollow cylinder. The reaction frame let into the receiving vessel initially forms, with the base plate of the receiving vessel, a reaction chamber that is open on one side, the open side being the top of the reaction frame, i.e. the side opposite the base plate. The bottom side of the reaction frame is then the side that faces toward the base plate of the receiving vessel. Accordingly, the top side of the frame is the side that is directed away from the base plate of the receiving vessel. It is also conceivable for the reaction frame to have a lattice or mesh structure in the region of an opening on the base plate.
The reaction frame can be removed from the receiving vessel at any time and transferred to another receiving vessel filled with fresh reaction liquid, without transferring large amounts of the contaminated reaction liquid. The old reaction liquid can then drain off through the opening in the bottom side of the frame. Such a modular construction of the device, comprising a reaction frame and a receiving vessel, enables a buffer to be changed easily. In particular, the device does not have to be tipped for changing the buffer.
The reaction frame may have a cover plate, which is fixedly or detachably connected to the top side of the reaction frame, wherein the cover plate may substantially completely cover the top side of the reaction frame and of the receiving vessel. A detachable connection may be achieved, for instance, by a plug connection. As soon as the cover plate covers the reaction frame and possibly the receiving vessel, the reaction chamber is completely closed. This prevents foreign bodies from entering the reaction chamber, or users of the device from being able to come into contact with the buffer during the electrophoresis.
According to a particular design, a horizontal reaction frame is provided. The horizontal reaction frame comprises four inner side walls, which are arranged in a cuboid shape in relation to one another. In this case, a corresponding receiving vessel may be provided, having four outer side walls that are likewise arranged in a cuboid shape in relation to one another. The inner side walls may each be of the same length or comprise two long and second short side walls. Correspondingly, the reaction frame may have a square or a rectangular shape. The same applies analogously to the outer side walls and to the receiving vessel. In the case of such an embodiment, the four outer side walls of the receiving vessel must have a common inner circumference that is larger than a common outer circumference of the inner side walls of the reaction frame. The inner and the outer side walls may each be glued to one another or plugged into one another. The outer side walls of the receiving vessel should be of a height that is at least half the height of the inner side walls of the reaction frame. Preferred, however, is an embodiment in which the outer side walls of the receiving vessel are at least as high as the inner side walls of the reaction frame. In addition, there should be a sufficiently large distance between the outer side walls of the receiving vessel and the inner side walls of the reaction frame such that the frame can be inserted into and removed from the tank quickly and easily. This distance should preferably be at least 0.5 cm.
A vertical embodiment may also be provided, which is characterized in that the reaction frame comprises an inner hollow cylinder. A corresponding receiving vessel is designed as an outer hollow cylinder, wherein the outer hollow cylinder of the receiving vessel has an inner circumference that is larger than an outer circumference of the inner hollow cylinder of the reaction frame. The outer hollow cylinder of the receiving vessel should be of a height that is at least half the height of the inner hollow cylinder of the reaction frame. Preferred, however, is an embodiment in which the outer hollow cylinder of the receiving vessel is at least as high as the inner hollow cylinder of the reaction frame. In addition, there should be a sufficiently large concentric distance between the inner and the outer hollow cylinder such that the frame can be inserted into and removed from the receiving vessel quickly and easily. This distance should preferably be at least 0.5 cm.
According to a preferred design, it is provided that the electrophoresis device has a first electrode and a second electrode. The first and the second electrode can be connected to a voltage source in order to generate an electric field. The biological tissue in this case is arranged substantially in the center of the reaction chamber because an approximately homogeneous electric field is concentrated there, between the electrodes arranged opposite one another. In particular, it is provided in this case that the first electrode and the second electrode are each realized as plate electrodes or as a grid electrode. Electrodes designed in this way have the advantage that the electric field is evenly distributed over the entire reaction chamber and is not just limited to a particular region within the reaction liquid. However, other electrode shapes are also conceivable, such as, for example, zigzag electrodes. It is also advantageous if the first electrode and the second electrode are in electrical contact with the power source, each via an electric leadthrough. The electric leadthroughs make it particularly easy to connect the electrodes to a voltage device.
It may be provided in this case that the electric contacts for connecting the leadthroughs to the power source are recessed into the cover plate of the reaction frame, such that the cover plate needs to be attached to, or placed on, the reaction frame in order to connect the device to the power source. In this case, the electric contacts can simultaneously serve as plug connections for fixing the cover to the reaction frame. This ensures that current can only flow in the reaction chamber when the cover is connected to the reaction frame, i.e. if there is a closed reaction chamber.
According to the horizontal embodiment, it is also provided that the first electrode is arranged, on the reaction chamber side, on one of the inner side walls of the reaction frame, and that the second electrode is arranged, on the reaction chamber side, on the inner side wall that is opposite the first electrode. The two electrodes should be arranged in parallel and lie in the same horizontal central axis. Such an arrangement of the electrodes results in a current flow in the horizontal direction. The sample in this case should be arranged between the electrodes.
According to the vertical embodiment, the first electrode should be arranged, on the reaction chamber side, on the cover plate of the reaction frame, and the second electrode should be arranged, on the reaction chamber side, on the base plate of the receiving vessel. The two electrodes in this case should lie in the same vertical central axis. Such an arrangement of the electrodes results in a current flow in the vertical direction. The sample in this case should be arranged between the electrodes.
It may also be provided that the electrophoresis device comprises a sample cassette in which the sample is fastened and with which the sample can be arranged in the reaction chamber, between the electrodes.
The sample cassette in this case may be standardized. The use of the sample cassettes according to the invention facilitates the execution of electrophoresis methods. It is possible, for example, for each cassette to have a bar code and/or color code by means of which the cassettes can be identified. Such a coding has the additional advantage that it indicates the orientation of the sample cassette relative to the direction of electrophoresis. In this way, when the sample cassette is changed to another receiving vessel, it can easily be inserted in the correct electrophoresis direction. Moreover, a cassette holder, into which the sample cassette can be clamped, may also be provided.
According to a preferred design, it is provided that the sample cassette comprises a base element and a cover element, which can be plugged together to form a cassette enclosing the sample, wherein the base element and the cover element are pivotably connected to one another, in particular via a flexible connection (for example a hinge). According to a preferred embodiment, it is provided that the sample cassette, or the cassette, is perforated, at least in sections, wherein in particular the base element and the cover element each have a multiplicity of perforations arranged in a grid-like manner. The perforations allow buffer to reach the tissue sample, which in turn ensures that the electric current can remove the desired substances from the sample. Such sample cassettes are particularly suitable for use in a clearing method because they can be easily standardized.
It may further be provided that the reaction frame has at least one receiving profile for receiving the sample cassette (or for receiving the cassette holder). According to the horizontal design of the device, grooves are provided for this purpose, which are each realized, on the reaction chamber side, on two opposite inner side walls and extend in the vertical direction. Advantageously, the at least one receiving profile may also comprise a horizontally realized groove, which is realized, on the reaction chamber side, in the base plate of the receiving vessel. According to this solution, the sample cassette can be inserted into the grooves by being slid-in in the vertical direction. A receiver realized in this way has the advantage that the sample cassette can be easily and reliably introduced into the receiver. Furthermore, as a result of this construction, the reaction chamber is divided into a first reaction compartment and a second reaction compartment when the sample cassette or the cassette holder is received in the receiving profile. In order that the sample cassette or the cassette holder can divide the reaction chamber into two reaction compartments, the sample cassette or the cassette holder is realized in such a manner that they each protrude above the surface of the reaction liquid when they are received in the receiving profile of the chamber. This ensures that the current that flows between the electrodes during the electrophoretic clearing method passes exclusively through the sample cassette, or the cassette holder, and in particular through the tissue sample.
According to an advantageous development, it is also provided, in the case of the horizontal embodiment of the electrophoresis device, that the receiving profile is arranged along a vertical central axis. It is also possible for the sample cassette and the receiving profile to be realized in such a manner that the biological sample is oriented substantially perpendicularly to the horizontal central axis, and thus parallel to the electrodes, when the sample cassette is received directly in the receiving profile. Correspondingly, the cassette holder should also be realized in such a manner that the biological sample is oriented substantially perpendicularly to the horizontal central axis when the cassette holder is received in the receiving profile. This allows the biological sample to be arranged in the reaction chamber where an approximately homogeneous electric field is concentrated between the electrodes. It is also conceivable that a buffer change can be performed in the reaction chamber by removing the sample cassette from the electrophoresis device in an upward direction and inserting it into a reaction chamber filled with fresh, or new, buffer. This can also simplify the changing of the buffer. The receiving profile should preferably have a locking mechanism by which the sample cassette, or the cassette holder, can be locked at a particular position relative to the reaction frame. This mechanism may be, for example, a tapering of the at least one groove in the direction of the bottom side of the reaction frame, which prevents the sample cassette, or the cassette holder, from sliding downward, i.e. in the direction of the bottom side, through the receiving profile of the reaction frame. The locking mechanism enables the reaction frame to be transferred quickly and safely from one receiving vessel to another without the risk of loss of the sample cassette, or the cassette holder.
According to the vertical embodiment, it may be provided that the receiving profile is an annular support, which is arranged, on the reaction chamber side, on the inner hollow cylinder and in which the sample cassette (or the cassette holder) is received. The reaction chamber in this case is divided into a first reaction compartment and a second reaction compartment when the sample cassette, or the cassette holder, is received in the receiving profile. To perform vertical electrophoresis, provision may also be made for the cassette holder or the receiving profile to have at least one vent hole that connects the two reaction compartments. The vent hole serves, when the reaction chamber is filled with buffer, to discharge upwardly gas or air bubbles produced in the lower part. The vent hole allows these bubbles to pass through so that they can reach the surface unhindered. In addition, the receiving profile may be inclined relative to the horizontal central axis. The inclination has the advantage that the cassette holder, or the sample cassette, can also be received with an inclination relative to the horizontal central axis. It is also conceivable in this case for the vent hole to be realized at the highest point of the cassette holder, or of the receiving profile, the highest point being understood to be that region of the cassette holder or the receiving profile that is closest to the surface of the reaction liquid. In this way, advantageously, the gas bubbles produced when the lower reaction chamber is filled collect in the vicinity of the vent hole, through which they can then be discharged toward the surface of the reaction liquid.
It may additionally be provided that the inner and outer side walls and the base plate are made from a chemically inert and electrically insulating material, in particular from glass or plastic. It is conceivable, for example, for the receiving vessel and the frame to be made of acrylic glass. The sample cassette and the cassette holder are also preferably made from a chemically inert and electrically insulating material, the sample cassette and the cassette holder preferably being made from a plastic, particularly preferably from polyoxymethylene. This ensures that the current that flows between the electrodes during the electrophoretic clearing process passes exclusively through the tissue sample and not through the sample cassette or the cassette holder.
According to the vertically realized electrophoresis device, the receiving vessel comprises a base plate and an outer hollow cylinder. The reaction frame comprises an inner hollow cylinder. Both hollow cylinders in this case are rotationally symmetrical about a vertical central axis. The arrangement may be characterized in that the outer circumference of the inner hollow cylinder is smaller than the inner circumference of the outer hollow cylinder, thereby creating an annular gap between the inner hollow cylinder and the outer hollow cylinder when the reaction frame is received in the receiving vessel. Such an electrophoresis device can be produced and assembled quickly and easily, and is therefore particularly easy to use.
It may further be provided, according to one embodiment, that the inner hollow cylinder is connected to the cover plate and extends vertically in the direction of the base plate. The outer hollow cylinder extends vertically from the base plate in the direction of the cover plate, a first height of the inner hollow cylinder being less than a second height of the outer hollow cylinder, thereby forming a gap between the base plate and the end of the inner hollow cylinder on the base plate side.
It may be provided that the first electrode is attached to the reaction frame, starting from the central horizontal axis, in the direction of the cover plate in the upper chamber element, namely on the side of the inner hollow cylinder that faces toward the reaction chamber. The distance between the first electrode and the central horizontal axis in this case should be greater than the distance between the first electrode and the cover plate. The second electrode, on the other hand, may either be attached concentrically in the receiving vessel, on a side of the base plate that faces toward the reaction chamber, or on a side of the outer hollow cylinder that faces toward the reaction chamber, or on a side of the inner hollow cylinder that faces toward the outer hollow cylinder. In the last two cases, a gap should be formed between the end of the inner hollow cylinder on the base plate side and the base plate. This can be achieved by use of a cover plate to which the inner hollow cylinder is attached. The inner hollow cylinder then extends vertically in the direction of the base plate, and may be of a first height that is less than the second height of the outer hollow cylinder. It is provided in particular that the second (lower) electrode is arranged slightly above the gap. Gas bubbles produced at the second electrode during electrophoresis rise due to the arrangement of the lower electrode in the annular space between the outer and inner hollow cylinder, and do not collect under the sample cassette, or under the cassette holder. In order that the gas bubbles produced in this way can escape from the electrophoresis device, the outer hollow cylinder may have, for example, perforations in a region near the cover plate, via which the gas bubbles can be released to the external environment. It may further be provided that the cover plate does not touch the outer hollow cylinder, such that a small gap remains between the outer hollow cylinder and the cover plate when the cover plate rests on the inner hollow cylinder. The gas bubbles can then escape to the outside through this gap. The first electrode and the second electrode may each be of an annular shape. This ensures that the electric field is evenly distributed over the entire reaction chamber. In this embodiment of the reaction chamber, the sample and the electrodes are arranged substantially in the same vertical plane. A vertical electrophoresis device has the advantage that the sample cassette, together with the tissue sample, can be arranged horizontally.
It may be provided that the cover plate has a vertical pin for closing and opening the vent hole of the cassette holder, or of the receiving profile, the vertical pin passing through a corresponding hole in the cover plate such that it can be operated from outside the reaction chamber. The vertical pin is provided in particular to selectively open or close the vent hole, such that air bubbles that collect in the lower reaction chamber under the sample cassette or cassette holder when it is filled with reaction liquid can escape through the vent hole when it is open. After the reaction chambers have been completely filled with reaction liquid and the air bubbles have escaped, the vent hole is closed by means of the vertical pin.
The cover plate may advantageously have a through-hole for filling the reaction chamber with reaction liquid, the through-hole preferably being closable. In addition, it is conceivable for the through-hole to be recessed substantially centrally into the cover plate. The electrophoresis device can be easily filled with reaction liquid, or buffer, via the through-hole in the cover plate. The possibility of filling the two reaction compartments separately, i.e. separately from each other, with reaction liquid is particularly gentle on the sample.
In the case of the vertical design, a buffer change can be realized by removing the inner hollow cylinder of the reaction frame, together with the sample, from the outer hollow cylinder of the receiving vessel and inserting it into a second receiving vessel filled with unused buffer. The reaction liquid can then drain off through the opening in the bottom side of the cylindrical frame. In particular, this enables the buffer to be changed quickly during the clearing process. As a result of the exchanging of the reaction liquid, the originally low electric current, which has increased due to the elution of substances from the tissue, is reduced again to the low initial value. The point in time at which the buffer has to be exchanged is indicated by the power supply, according to the current-voltage characteristic.
A further embodiment provides that the first electrode has a first electric leadthrough for contacting to the power source, and that the second electrode has a second electric leadthrough for contacting to the power source. Advantageously, the first leadthrough to the power source may be arranged in the cover plate, and the second leadthrough in the cover plate or base plate.
It is additionally conceivable for the inner and the outer hollow cylinder and the base plate to be made from a chemically inert and electrically insulating material, in particular from glass or from a plastic (e.g. acrylic glass). It is further provided that the sample cassette and the cassette holder are made from a chemically inert and electrically insulating material, the sample cassette and the cassette holder preferably being made from a plastic, particularly preferably from polyoxymethylene. Finally, it is also conceivable for the sample cassette to be perforated, at least in sections, the base element and the cover element in particular each having a multiplicity of perforations arranged in a grid-like manner. This ensures that the current that flows between the electrodes during the electrophoretic clearing process passes through the tissue sample and not through the sample cassette or the cassette holder. A current flow past the sample, for instance through the reaction liquid or through sections of the sample cassette or the cassette holder, is to be avoided. It may further be provided that the sample cassette comprises a base element and a cover element that can be plugged together to form a cassette enclosing the sample, the base element and the cover element being pivotably connected to one another, in particular via a flexible connection or hinge.
Further features, details and advantages of the invention are given by the wording of the claims and by the following description of exemplary embodiments, with reference to the drawings.
The reaction frame 3 shown in
It may be provided in this case that the grooves 33 taper toward the bottom side 5 of the reaction frame 3, thereby preventing the sample cassette 19, or the cassette holder 20, from sliding downward (i.e. toward the bottom side 5) through the grooves 33 of the reaction frame 3. The locking mechanism thus enables the reaction frame 3 to be transferred quickly and safely from one receiving vessel 7 to another without the risk of loss of the sample cassette 19, or of the cassette holder 20. A receptacle profile 24 realized in such a manner thus has the advantage that the sample cassette 19, and the cassette holder 20, can be easily and reliably inserted into the receiver 24.
The sample cassette 19 is inserted into the grooves 33 as shown in
According to the design shown, the first electrode 11 and the second electrode 12 are each realized in the form of a rod electrode. However, plate electrodes 11, 12 or electrodes 11, 12 realized as a grid may also be provided. Plate electrodes 11, 12 have the advantage that the electric field is evenly distributed over the entire reaction chamber 9 and is not just limited to a particular region within the reaction liquid 27. It is further advantageous if the first electrode 11 and the second electrode 12 are each in electrical contact with the power source via an electric leadthrough 36, 37. The electric leadthroughs 36, 37 make it particularly easy to connect the electrodes 11, 12 to a voltage device.
As can also be seen from
Additionally shown in
A vertical electrophoresis device 1 according to
The design shown in
Alternatively, it is possible to fill the two reaction compartments 25, 26 separately, i.e. separately from each other, with reaction liquid 27. For this purpose, the cover plate 10 has a through-hole 30 for filling the upper reaction compartment 25 with reaction liquid 27. The through-hole 30 is centrally recessed in the cover plate 10 and realized in principle so to be closable. However, it should be open during the electrophoresis process so that the gas produced at the electrodes 11, 12 can escape. Preferably, the sample cassette 19 or the cassette holder 20 has a vent hole 28, so that the gas 34 produced at the lower electrode 12 (
The first electrode 11 is attached to an end region of the inner hollow cylinder 17 near the top side 4, or the cover plate 10, namely on the side of the inner hollow cylinder 17 that faces toward the reaction chamber 9. According to
It is additionally provided that the cassette holder 20 has a vent hole 28 (
The invention is not limited to any of the above-described embodiments, but may be varied in a variety of ways.
All of the features and advantages, including constructional details, spatial arrangements and method steps given by the claims, the description and the drawing, can be essential to the invention both individually and in the widest variety of combinations.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2020 109 087.0 | Apr 2020 | DE | national |
This is the United States National Phase of International Patent Application No. PCT/EP2021/057619, filed Mar. 24, 2021, which claims priority to German Application No. DE 10 2020 109 087.0, filed Apr. 1, 2020, the entire contents of each of which incorporated by reference herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/057619 | 3/24/2021 | WO |
