Cryowriting system

Abstract

System (1) for applying a sample solution and / or an auxiliary solution to a carrier device (5), the system (1) comprising a dosing device (3) for dosing the sample solution and / or for dosing the auxiliary solution, an application device (4) connected to the dosing device (3) with which the dosed sample solution and / or the dosed auxiliary solution can be applied to the carrier device (5), and a guide device (2) for the application device (4), with which the application device (4) is movable relative to the carrier device (5), characterized in that the system (1) is configured to apply at least one preparatory layer of the sample solution or of the auxiliary solution to the carrier device (5) and then to apply at least one regular layer of the sample solution over the at least one preparatory layer of the sample solution or of the auxiliary solution, wherein the at least one preparatory layer is adapted to counteract destruction and / or clumping of the sample substance from the at least one regular layer.

Description

Technical FieldThe invention relates to a system for cryowriting according to the preamble of independent claim 1.Such a system is generally used for preparing a protein sample on a support structure for cryo-electron microscopy (cryo-EM). The carrier structure is in this case regularly designed in the form of a grid for receiving the protein sample (such as, in particular, for example, a so-called cryo-EM grid), which is applied to the cryo-EM grid by a dosing head of the system. The system also regularly comprises a temperature-controlled stage which is designed to maintain the cryo-EM grid at a predefined temperature when the cryo-EM grid is arranged thereon. The system also generally includes a transfer mechanism configured to place the cryo-EM grid from the temperature controlled stage into a container containing a liquid cryogen (e.g., ethane) such that the sample on the cryo-grid contacts the cryogen for the purpose of vitrifying.In general, thin vitrified sample layers are desirable for the corresponding electron microscopy analysis, the proteins to be investigated being to be obtained as well as possible.For this purpose, it is advisable that the corresponding protein solution can be applied to the cryo-EM grid with good resolution, the protein solution is or remains cooled as well as possible when applied to the cryo-EM grid, and the cryo-EM grid remains cooled as well as possible even during transfer into the cryogenic container.In this respect, various solutions have already been proposed, which will be described below.Prior ArtThus, WO 2024 / 184 341 A1 discloses a syringe pump (i.e. in particular a picopump) which comprises a syringe unit having a cylinder and a piston and a first piston drive which has a spindle structure and a motor. The cylinder has a hollow interior and an outlet in fluid communication with the hollow interior, the piston extending into the hollow interior of the cylinder and defining a chamber within the cylinder. The spindle structure of the first piston drive is configured to convert a rotational movement of the motor into a translatory movement, wherein the spindle structure of the first piston drive is coupled to the piston of the syringe unit, such that the translatory movement generated by the spindle structure moves the piston relative to the cylinder and thereby changes a volume of the chamber in the interior of the cylinder. A second piston drive is provided, which includes a piezoelectric actuator coupled to the piston to move the piston relative to the cylinder and thereby change the volume of the chamber inside the cylinder. With this syringe pump, comparatively high volumes of liquid can be provided with high precision or nominal resolution.In WO 2025 / 036 907 A1 a method is described for cooling a sample solution in a pipette configured for preparing a sample for electron microscopy, the method comprising the steps of: cooling a pipette arranged in a pipette holding unit to a target temperature in a range from about -2°C to about the dew point temperature; cooling a cooling liquid and / or a sample solution to a target temperature in the range from about 0°C to about the dew point temperature; aspirating cooling liquid with the pipette; aspirating sample solution with the pipette; and preparing a sample for electron microscopy on a sample carrier structure preferably cooled to about the dew point temperature.WO 2024 / 061 904 A1 relates to a gripper unit for gripping and transferring carrier structures, in particular gratings or cryo-EM grids for electron microscopy. The gripper unit comprises gripping fingers configured to grip and release the support structure, the gripping fingers being made of a thermally conductive material. The gripper unit further comprises a temperature control element configured to set the temperature of the gripping fingers to a predetermined value. Further described is a corresponding transfer system that transfers cryo-EM grids from a substantially horizontal position to a substantially vertical position to quickly insert the cryo-EM grid into the cryogen. In order to protect the thin sample layer even better during this transfer, the gripper has a shielding compartment into which the cryo-EM grid is inserted.The above solutions have already led in practice to significantly improved results in cryo-writing.However, the problem still arises in the production of thin, vitrified sample layers for cryo-EM analysis that most target proteins interact with the air-water interface (also referred to as AWI or air-water interface), which leads to partial or complete denaturation and / or preferential alignment of the proteins at the air-water interface, which seriously impairs the quality of the data obtained. In addition, proteins often aggregate when mixed with activators / inhibitors, making structural analysis of the corresponding complex, including important protein-drug complexes, difficult. Finally, analysis of conformational changes caused by the formation of a protein complex or during enzymatic / functional cycles in time-resolved studies requires precise mixing on the grid immediately prior to the vitrification.The present invention is therefore based on the object of proposing a system with which the aforementioned problems can be solved.SUMMARY OF THE INVENTIONThe object is achieved according to the invention by a system as defined in independent claim 1. Advantageous embodiments of the invention are evident from the dependent claims.The essence of the invention consists in the following: a system for applying a sample solution and / or an auxiliary solution to a carrier device, wherein the system comprises a dosing device for dosing the sample solution and / or for dosing the auxiliary solution, an application device connected to the dosing device, with which the dosed sample solution and / or the dosed auxiliary solution can be applied to the carrier device, as well as a guide device for the application device, with which the application device is movable relative to the carrier device. In addition, the system is configured to apply at least one preparatory layer of the sample solution or of the auxiliary solution to the carrier device and subsequently to apply at least one regular layer of the sample solution via the at least one preparatory layer of the sample solution or of the auxiliary solution (wherein the sequence can also be reversed). The at least one preparatory layer is adapted to counteract destruction and / or agglomeration of the sample substance (generally proteins) from the at least one regular layer.The term "auxiliary solution" is to be understood in the present case as meaning that it does not contain the substance relevant for later cryo-electron microscopy or relevant amount of sample substance.The corresponding "preparatory layer" thus either does not comprise a sample or protein solution or, if it comprises a sample or protein solution, it is applied to the cryo-EM grid only so thinly that the lattice clearances do not remain wetted by sample or protein solution, so that only the carbon film of the grid is saturated with sample or protein solution.The term "regular layer" comprises applying sample or protein solution to the preparatory layer located below (as a rule) in such a quantity that the protein molecules to be investigated in the solution optimally arrange themselves within the lattice interstices of the cryo-EM grid (i.e. the lattice interstices remain wetted with the sample or protein solution).Preferably, the carrier device comprises a cryo-EM grid, wherein the cryo-EM grid is preferably arranged on a cooling unit or a cooling plate. The cryo-EM grid is preferably a flat carbon support grid on copper or gold, which is optimized for cryo-electron microscopy. Cooling is necessary in that most proteins are not stable when extracted from their natural environment. Membrane proteins are particularly susceptible to denaturation when extracted from the lipid bilayer by detergents. Solubilization, however, is a prerequisite for the purification and cryo-electron microscopy of membrane proteins. Such solutions to be cooled or kept cooled helps stabilize the membrane proteins. In this regard, reference is made to the corresponding disclosure from WO 2025 / 036 907 A1.Preferably, the dosing device comprises at least one picopump. In this way, the required small amounts of sample solution or of auxiliary solution can be provided for the application device. The metering device is preferably configured to provide or convey a volume of the sample solution to be applied to the carrier device or a volume of the auxiliary solution to be applied to the carrier device in a range of 1 nanoliter or less. With regard to a possible configuration of such a picopump, reference is made to the corresponding disclosure in WO 2024 / 184 341 A1.Preferably, the application device comprises a pipette or a pin. Both devices are suitable for the desired application of the sample solution or of the auxiliary solution in the nanoliter range to the cryo-EM grid. Preferably, the application device is also cooled or kept cool (in a corresponding holder), as described, for example, in WO 2025 / 036 907 A1.Preferably, the guiding device comprises a high precision transport device, such as a robot arm. The guide device operates three-dimensionally and accurately in the micrometer range. The guide device moves the pipette or the pin along the grid surface of the cryo-EM grid while the dosing device or the picopump delivers the sample liquid. In this way, any pattern (spirals, lines, crossed lines) can be written, the layer thickness being determined by the speed of the pipette, its diameter and the dispensing rate. Such layers can be applied within 1 second.Preferably, the at least one preparatory layer is applied at a concentration below the critical micellar formation concentration. In this way, the AWI problem can be eliminated. The first or preparatory layer is written with the corresponding auxiliary solution (i.e. a detergent) at a concentration regularly far below the critical micellar concentration (CMC or critical micellar concentration). Subsequently, while the second or regular layer is written with the protein solution, the detergent with a molecular weight of about 0.5 kDa (kilodalton) diffuses within 1 ms to the AWI where it is trapped, while typical proteins with a mass between 50 kDa and 1MDa move about ten times more slowly. Thus, with this dual or multilayer writing technique, the AWI problems can be solved by the detergent modulating the AWI before proteins can interact with the AWI. Since the detergent is below the CMC and moves to the AWI within about one millisecond, adverse interactions with the protein are virtually impossible.Preferably, the at least one preparatory layer (or the detergent used as an auxiliary solution thereof) comprises a surfactant such as PNVP (poly(N-vinylpyrrolidone)). This has proven to be particularly effective in practice for the multilayer cryowriting described in the present case.In another preferred embodiment, the at least one preparatory layer comprises a thin layer of the sample solution. As already explained above, this serves the purpose that the lattice clearances of the cryo-EM grid do not remain wetted with sample or protein solution, but that the protein molecules present saturate the carbon lattice before the application of the regular layer.Preferably, the guiding device and the application device are further configured to apply the volume of the sample solution or the volume of the auxiliary solution to the carrier device in less than one second in a regular layer or in a preparatory layer.Finally, any desired pattern (spirals, lines, crossed lines) can be written in this way, wherein the layer thickness is determined by the speed of the pipette, its diameter and the dispensing rate.In other words, the protein aggregation when mixed with activators / inhibitors can also be reduced in particular by writing in a plurality of (protein-containing) layers. The activator / inhibitor solution is applied as the first (preparatory) layer and the protein interacting with these compounds is regularly applied as the second (regular) layer. The diffusion times for small molecules (<300 Da) are usually three orders of magnitude shorter than when writing the second layer. Thus, the intermixing on the grid will be complete prior to glazing. Note that protein aggregation is not expected in the short time of writing the second layer.In the investigation of conformational changes of proteins which join to form a protein complex, fundamentally different time scales must be taken into account. Proteins can undergo large global conformational changes occurring on long time scales (milliseconds to seconds). In the slower processes, writing in multiple layers thus opens up new experimental approaches.In a further embodiment, the invention also includes: (embodiment 1) a method, in particular a cryowriting method, for applying a sample solution and / or an auxiliary solution to a carrier device, wherein the method comprises the following steps: (a) providing a carrier device and a sample solution (i.e. in particular a protein solution) and / or an auxiliary solution; (b) applying at least one preparatory layer of the sample solution or of the auxiliary solution to the carrier device; (c) applying at least one regular layer of the sample solution via the at least one preparatory layer of the sample solution or of the auxiliary solution; wherein the at least one preparatory layer is adapted to counteract destruction and / or agglomeration of the sample substance from the at least one regular layer. Steps (b) and (c) can be carried out in the sequence mentioned above in terms of time or else in the reverse sequence, i.e. step (c) can also be carried out before step (b), i.e. it is also conceivable for the (regular) second layer to be applied before the first (preparatory) layer.(Embodiment 2) The method according to Embodiment 1, wherein a cryo-EM grid is provided as the carrier device, wherein the cryo-EM grid is preferably arranged on a cooling unit.(Embodiment 3) Method according to Embodiment 1 or 2, wherein a device for metering the sample solution and / or the auxiliary solution comprises at least one picopump. The metering device is usually connected to the application device, with which the metered sample solution and / or the metered auxiliary solution is applied to the carrier device.(Embodiment 4) Method according to one of Embodiments 1 to 3, wherein a pipette or a pin is provided as application device.(Embodiment 5) Method according to one of Embodiments 1 to 4, wherein a high-precision transport device, preferably a robot arm, is provided as a guide device for the application device. With the guide device, the application device is moved (three-dimensionally and micrometer-accurate) relative to the carrier device.(Configuration 6) Method according to one of Configurations 1 to 5, wherein the at least one preparatory layer according to step (b) is applied at a concentration below the critical micellar formation concentration.(Embodiment 7) The method according to any one of Embodiments 1 to 6, wherein as the at least one preparatory layer, a surfactant such as PNVP (poly(N-vinylpyrrolidone)) is provided.(Embodiment 8) The method according to any one of Embodiments 1 to 5, wherein a thin layer of the sample solution is provided as the at least one preparatory layer.(Embodiment 9) The method according to any one of Embodiments 1 to 8, wherein a volume of the sample solution to be applied to the carrier device or a volume of the auxiliary solution to be applied to the carrier device is provided in a range of 1 about nanoliter or less (i.e., from the metering device to the application device).(Embodiment 10) The method according to any one of Embodiments 1 to 9, wherein the volume of the sample solution or the volume of the auxiliary solution is applied to the support device as a regular layer or as a preparatory layer in less than about one second.Brief Description of the DrawingsFurther advantageous embodiments of the invention are evident from the following description of exemplary embodiments of the invention with the aid of the schematic drawing. In particular, a system according to the invention is described below with reference to the attached drawings on the basis of an exemplary embodiment. The following are shown: FIG. 1 is a schematic view of a system according to the invention with its essential components; FIG. 2 : shows a schematic view of a carrier device (multilayer application) described with the system according to the invention; FIG. 3 : a cryo-EM recording of a grid with proteins which are well distributed in the grid free space; and FIG. 4 : a cryo-EM image of a grid with proteins, which are predominantly arranged on the carbon film.Way(s) for Carrying Out the InventionCertain terms may be used in the following description for convenience and are not intended to be limiting. The words "right", "left", "lower" and "upper" denote directions in the drawing to which reference is made. The terms "inwardly", "outwardly", "below", "above", "left", "right" or the like are used to describe the arrangement of designated parts relative to each other, the movement of designated parts relative to each other, and the directions toward or away from the geometric center of the invention, as well as designated parts thereof, as illustrated in the figures. These spatial relative indications also comprise different positions and alignments than those illustrated in the figures. For example, when a part illustrated in the figures is turned over, elements or features described as "below" are then "above.". The terminology includes the words expressly mentioned above, derivatives thereof, and words of similar import.In order to avoid repetitions in the figures and the associated description of the various aspects and exemplary embodiments, certain features should be understood as common for various aspects and exemplary embodiments. The omission of an aspect in the description or a figure does not suggest that this aspect is missing in the associated exemplary embodiment. Rather, such omission may serve for clarity and prevention of repetitions. In this context, the following definition applies to the entire further description: If reference numerals are contained in a figure for the purpose of graphic uniqueness, but are not mentioned in the directly associated text of the description, reference is made to their explanation in the preceding descriptions of the figures. If reference numerals are also mentioned in the description text directly belonging to a figure, which are not contained in the associated figure, reference is made to the preceding and following figures. Like reference numerals in two or more figures represent like or like elements.FIG. 1 schematically illustrates a system 1 according to the invention for multilayer cryowriting. The system comprises a base 7 from which a guide device 2 extends having at least an arm portion 2a and a head portion 2b. On the base 8, an input unit 8 such as a PC and a controller are disposed. However, the input unit 8 and / or the controller can also be wirelessly connected to the system 1, i.e. in the form of a hand-held device, such as a tablet or a mobile telephone. On and / or in the head section 2 b, the dosing device 3 is arranged, which delivers the respective volumes of liquid (i.e. protein solution or detergent) to the application device 4, here in the form of a pipette. To apply the desired layers, the pipette 4 is moved by means of the guide device 2 relative to the carrier device arranged on the cooling unit 6, here in the form of a cryo-EM grid. The guide device 2 moves in three-dimensional space, as indicated by the triaxial coordinate system (X, Y, Z). The guide device 2 is usually designed as a high-precision transport device which operates with precision by micrometers. The pipette 4 (or an alternative pin) can also be kept cooled by means of a corresponding holder, i.e. as described for instance in WO 2025 / 036 907 A1.FIG. 2 illustrates a cryo-EM grid 5 with a multilayer 9 according to the invention in the form of a worm. The second and optionally the subsequent layers can be applied from the starting point of the first screw shape over the same or they can be applied from the end point of the first screw shape over the same (i.e. backwards). This naturally applies to all conceivable multilayer shapes.FIG. 3 is a cryo-EM image of a grid with proteins, which are distributed particularly well in the grid clearance of the grid by means of the multilayer application according to the invention.FIG. 4, in contrast, shows a cryo-EM image of a grid with proteins without the multilayer application according to the invention. Here it can clearly be seen that the proteins accumulate (in an undesirable manner) predominantly on the carbon film of the grid.Although the invention has been illustrated and described in detail by means of the figures and the associated description, this illustration and this detailed description are to be understood as illustrative and exemplary and not as restrictive of the invention. In order not to clarify the invention, in certain instances, well-known structures and techniques may not be shown and described in detail. It is to be understood that changes and modifications may be made by those skilled in the art without departing from the scope of the following claims. In particular, the present invention covers further embodiments with any combinations of features that may deviate from the explicitly described combinations of features.The present disclosure also encompasses embodiments having any combination of features mentioned or shown above or below with respect to various embodiments. It likewise comprises individual features in the figures, even if they are shown there in connection with other features and / or are not mentioned above or below. The alternatives of embodiments described in the figures and the description and individual alternatives of the features thereof can also be excluded from the subject matter of the invention or from the disclosed subject matter. The disclosure comprises embodiments which comprise exclusively the features described in the claims or in the exemplary embodiments and also those which comprise additional other features.Furthermore, the term "comprise" and derivatives thereof does not exclude other elements or steps. Likewise, the indefinite article "a" and derivatives thereof does not exclude a multiplicity. The functions of a plurality of features listed in the claims can be fulfilled by a unit or a step. The mere fact that certain masses are recited in mutually different dependent claims does not mean that a combination of these masses cannot be used advantageously. The terms "substantially", "approximately", "approximately" and the like in connection with a property or a value also define, in particular, exactly the property or exactly the value. The terms "about" and "about" in the context of a given numerical value or range may refer to a value or range that is within 20%, within 10%, within 5%, or within 2% of the given value or range. All reference numerals in the claims should not be understood as limiting the scope of the claims.List of Reference Numerals:1 System 2 Guide device 2 a Armabschnitt portion 2 b Kopfabschnitt portion 3 Metering device 4 Application device 5 Carrier device 6 Cooling unit 7 Base 8 Input unit 9 Multilayer (exemplary) Stretch ControllerReferences included in the specificationThis list of documents cited by the applicant has been produced in an automated manner and is only included for the better information of the reader. The list is not part of the German patent application or utility model application. The DPMA does not take any adhesion for any faults or omissions.Patent Literature citedWO 2024 / 184 341 A1 [0006, 0018]WO 2025 / 036 907 A1 [0007, 0017, 0019, 0041]WO 2024 / 061 904 A1

[0008]

Claims

System (1) for applying a sample solution and / or an auxiliary solution to a carrier device (5), wherein the system (1) comprises a metering device (3) for metering the sample solution and / or for metering the auxiliary solution, an application device (4) connected to the metering device (3) with which the metered sample solution and / or the metered auxiliary solution can be applied to the carrier device (5), and a guide device (2) for the application device (4) with which the application device (4) is movable relative to the carrier device (5), characterized in that the system (1) is configured, at least one preparatory layer of the sample solution or of the auxiliary solution to be applied to the carrier device (5) and subsequently at least one regular layer of the sample solution to be applied via the at least one preparatory layer of the sample solution or of the auxiliary solution, wherein the at least one preparatory layer is adapted to counteract destruction and / or agglomeration of the sample substance from the at least one regular layer.The system (1) of claim 1, wherein the support device (5) comprises a cryo-EM grid, wherein the cryo-EM grid is preferably disposed on a cooling unit.The system (1) according to claim 1 or 2, wherein the dosing device (3) comprises at least one picopump.The system (1) according to any one of claims 1 to 3, wherein the applicator (4) comprises a pipette or a pin.The system (1) according to any one of claims 1 to 4, wherein the guiding device (2) comprises a high precision transport device, preferably a robot arm.The system (1) according to any one of claims 1 to 5, wherein the at least one preparatory layer is deposited at a concentration below the critical micellizing concentration.The system (1) according to any one of claims 1 to 6, wherein the at least one preparatory layer comprises a surfactant such as PNVP (poly(N-vinylpyrrolidone)).The system (1) according to any one of claims 1 to 5, wherein the at least one preparatory layer comprises a thin layer of the sample solution.The system (1) according to any one of claims 1 to 8, wherein the dosing device (3) is configured to provide a volume of the sample solution to be applied to the carrier device (5) or a volume of the auxiliary solution to be applied to the carrier device (5) in a range of 1 about nanoliter or less.The system (1) according to any one of claims 1 to 9, wherein the guiding device (2) and the application device (4) are configured to apply the volume of the sample solution or the volume of the auxiliary solution to the carrier device (5) in less than about one second in a regular layer or in a preparatory layer.

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