Method for cooling a sample solution in a pipette, pipette holding and cooling unit, and cooling plate

Abstract

The present invention relates to a method for cooling a sample solution in a pipette configured for preparing a sample for electron microscopy, the method comprising the following steps: (a) A step of cooling the pipette (5) positioned within the pipette holding unit (1) to a target temperature (T1) in the range of approximately -2°C to approximately the dew point temperature; (b) A step of cooling the coolant and / or sample solution to a target temperature (T2) in the range of approximately 0°C to approximately the dew point temperature; (c) Step of drawing up the coolant using a pipette (5); (d) A step of aspirating the sample solution with a pipette (5); and (e) A step of preparing a sample for electron microscopy on a sample support structure, wherein the sample support structure is preferably cooled to approximately the dew point temperature. The present invention also relates to a pipette holding and cooling unit (1) and a cooling plate (6).

Description

Technical Field

[0001] The present invention relates to a method for cooling a sample solution in a pipette configured to prepare a sample for electron microscopy, a pipette holding and cooling unit for cooling such a pipette, a cooling unit for cooling a coolant and / or a sample solution, and a system for performing the method.

Background Art

[0002] As a novel method for preparing a sample for electron microscopy, cryo-writing is becoming increasingly important. Cryo-writing is particularly used, for example, using an aqueous protein sample solution, to write and / or prepare a cryo-grid for cryo-electron microscopy with each pipette. Thereby, the pipette used for cryo-writing sucks the aqueous protein solution from a suitable container and discharges the aqueous solution when writing a pattern on the grid. Such a technique is the subject of International Publication No. 2017 / 005297 (WO 2017 / 005297 A1).

[0003] Thus, lines, circles, or any combination thereof are written to create a sample layer, which is vitrified by immersing the grid, for example, in liquid ethane immediately after writing. The writing speed, pipette diameter, and pump rate determine the average thickness of the sample layer, which is typically between 1 and 2 μm.

[0004] The thickness of the layer is important in order to be able to determine the atomic structure of the protein or protein complex. If the layer is too thin, the protein will denature at the air / water interface of the water layer. If the layer is too thick, the protein will not generate sufficient image contrast when irradiated with an electron beam compared to the surrounding thick water layer. Thus, generating a layer of appropriate thickness is a major goal of all sample vitrification protocols for cryo-electron microscopy.

[0005]

[0005] The μm precision of the pump device used for cryo-writing (e.g., the pico-pump described in the applicant's Swiss patent application CH 000225 / 2023 (pre-publication)) and the movement of the pipette are important prerequisites for reproducibly writing sample layers of selected thickness.

[0006] Controlling sample evaporation is also an important topic. In an environment with 100% relative humidity, evaporation is minimal, and at high relative humidity and / or when the sample layer is close to the dew point temperature, evaporation is suppressed. The latter is the subject of international publication 2018 / 073242 (WO 2018 / 073242 A1).

[0007] While the grid itself can be cooled by a grid holder (for example, the gripper described in the applicant's Swiss patent application 001089 / 2022 (pre-publication: CH 001089 / 2022)), the heat of the applied sample solution needs to be transferred to a heat sink represented by the grid structure.

[0008] Typically, considering a grid geometry with a 10-20 nm thick carbon layer spanning grid squares with side lengths of 80 μm, it becomes clear that a sample layer 100 times thicker will not reach the dew point before significant evaporation occurs. On the other hand, if the sample solution and grid are maintained at the dew point, no evaporation occurs, and the vitrified layer may be too thick for (cryo) electron microscopy.

[0009] Many proteins are unstable when extracted from their natural environment. Membrane proteins, in particular, are prone to denaturation when extracted from the lipid bilayer with washing agents. However, solubilization is a prerequisite for the purification of membrane proteins and cryo-electron microscopy. Keeping such solutions cold helps stabilize membrane proteins.

[0010]

[0010] Therefore, an object of the present invention is to provide a method for cooling a protein sample solution and a pipette by means that can reproducibly ensure an appropriate sample layer thickness, and each pipette holding and cooling unit and each cooling plate should also be provided. [Overview of the project]

[0011] According to the present invention, these needs are solved by a method for cooling a sample solution in a pipette, as defined by independent claim 1; a pipette holding and cooling unit, as defined by independent claim 8; and a cooling plate, as defined by independent claim 17. Preferred embodiments are subject to the dependent claims.

[0012] In one embodiment, the present invention relates to a method for cooling a sample solution in a pipette configured for preparing a sample for electron microscopy, the method comprising the following steps: (a) A step of cooling a pipette, which is positioned and configured within a pipette holding and cooling unit, to a target temperature T1 in the range of approximately -2°C to approximately the dew point temperature; (b) A step of cooling the coolant and / or sample solution to a target temperature T2 in the range of approximately 0°C to approximately the dew point temperature; (c) Step of drawing up the coolant using a pipette; (d) A step of aspirating the sample solution with a pipette; and (e) A step of preparing (writing) a sample for electron microscopy on a sample support structure, wherein the sample support structure is preferably cooled to approximately the dew point temperature. Includes.

[0013] The advantage of the method according to the present invention is that the temperature of the deposit sample solution can be precisely controlled by the gripper (as in the case of the gripper described above as an application by the applicant) and / or by the clamp that holds the grid for writing, in relation to the sample support structure which is cooled to approximately the dew point temperature, thereby reducing the temperature offset of the grid temperature from the dew point temperature and the time delay between, for example, writing and insertion into liquid ethane. dThis allows for adjustment of the layer thickness. Therefore, the temperature offset should generally not be too large in order to achieve sufficiently accurate layer thickness control. However, if the pipette and sample solution (and coolant) are not properly cooled, the sediment sample may still evaporate too quickly. Thus, only with this "cold writing" method can a sample layer of a reproducible, predetermined thickness be reliably created, and fragile proteins and their complexes be reliably stabilized.

[0014] Generally, in an atmosphere with relatively low humidity and a relatively low dew point (e.g., winter time), evaporation of the sediment sample solution occurs relatively frequently (which can denature proteins), whereas in an atmosphere with relatively high humidity and a relatively high dew point (e.g., summer time), evaporation of the sediment sample solution does not occur relatively frequently (which may result in the sediment sample solution layer remaining too thick to ensure sufficient resolution under a microscope).

[0015]

[0015] The term “sample solution” as used herein relates to soluble protein complexes, but also to protein solutions in which membrane proteins are to be purified for electron microscopy examination.

[0016] The term "pipette" as used herein specifically refers to a micropipette, which is manufactured from a glass capillary combined with a pico-pump for dispensing liquids. Pipettes can be used in conjunction with a pipette holding unit, which allows for cooling of the pipette.

[0017] As used here, the term "electron microscopy" specifically refers to cryo-electron microscopy (cryo-EM).

[0018] The dew point temperature is the temperature of air required to cool (at a constant pressure) to achieve 100% relative humidity (RH). At this dew point, air can no longer hold any more moisture in gaseous form. The dew point is affected by the humidity and pressure of the air. The more moisture the air contains, the higher the dew point.

[0019] As used here, the "sample support structure" typically takes the form of a cryo-grid and / or cryo-EM grid, which usually has a mesh-like geometric shape coated with a perforated carbon film with a thickness of 10 nm to 20 nm.

[0020]

[0020] Preferably, for a pipette configured to be placed in a pipette holding unit, the target temperature T1 is between approximately -1°C and approximately the dew point temperature, preferably between approximately 0°C and approximately the dew point temperature. These temperature ranges have been proven to yield the best results, particularly for the purpose of cryo-writing.

[0021] Preferably, the target temperature T2 for cooling the liquid and / or sample solution is between approximately 0°C and approximately 5°C, preferably between approximately 0°C and approximately 3°C. In this way, the pipette is cooled from the inside, further improving the efficiency of layer thickness adjustment for the cryo-lighting procedure, thereby enabling electron microscopy with very good resolution.

[0022] Preferably, the pipette is configured to prepare a sample on the sample support structure with a thickness of approximately 500 nm to approximately 3000 nm, preferably approximately 1000 nm to approximately 2000 nm, and more preferably approximately 1250 nm to approximately 1750 nm. In this way, optimal preconditions for the cooling and cryo-lighting process can be ensured.

[0023] Preferably, the sample support structure is a cryo-grid. In a particularly preferred embodiment, the cryo-grid comprises a carbon layer and a gold layer. The additional gold layer supports the maintenance of the desired grid temperature and / or the desired deposited sample temperature.

[0024] Preferably, after step (e) and before the sample on the support structure is vitrified, a predetermined delay time t d is maintained. This delay prior to vitrification (i.e., plunging the sample support structure and / or grid into liquid ethane) after writing is also effective for adjusting the layer thickness of the sample solution deposited on the sample support structure and / or grid. The optimal delay time depends on the initial layer thickness and temperature of the deposited sample solution, and the temperature of the sample support structure and / or grid, respectively.

[0025]

[0025] In a further aspect, the present invention relates to a pipette holding and cooling unit that provides cooling to a pipette. The pipette holding and cooling unit includes a pipette configured to prepare a sample for electron microscopy, a body configured to receive the pipette therein, and a cooling unit configured to provide cooling to the body. The cooling unit is configured to cool the pipette received within the body to a target temperature T1 in the range of approximately -2°C to approximately the dew point temperature.

[0026] Preferably, the target temperature T1 is between approximately -1°C and approximately the dew point temperature, preferably between approximately 0°C and approximately the dew point temperature. These temperature ranges have been proven to provide the best results, particularly for the purpose of cryo-writing.

[0027] Preferably, the cooling unit comprises a cooling element and a cooling block, and the cooling element is in a heat transfer relationship with the body and the cooling block. Particularly preferably, the cold side of the cooling element abuts the body, and the warm side of the cooling element abuts the cooling block in order to dissipate the heat generated by the cooling element. In this way, a particularly accurate and uniform cooling effect can be achieved.

[0028] Preferably, the cooling element is in the form of a Peltier element. In a further preferred embodiment, the cooling block is a metal block, which is preferably a metal block through which a coolant (e.g., water) flows. This makes it possible to achieve ideal heat transfer characteristics.

[0029] Preferably, the pipette is configured to prepare a sample with a thickness of approximately 500 nm to approximately 3000 nm, preferably about 1000 nm to approximately 2000 nm, and more preferably about 1250 nm to approximately 1750 nm, on the sample structure. In this way, optimal preconditions for the cooling and cryo-lighting process can be ensured.

[0030]

[0030] Preferably, the pipette holding and cooling unit includes a clamp portion for holding the pipette, and the clamp portion is configured to be detachably attached to the main body. This clamp unit allows for easy replacement of the pipette without requiring additional equipment.

[0031] Preferably, the body of the pipette holding and cooling unit includes a hole configured to receive a clamp portion. The clamp portion advantageously includes a flexible outer periphery and a centrally positioned insertion projection. The insertion projection is introduced into the hole of the body at its distal end, and the outer periphery is pressed against the distal end of the body, thereby receiving the insertion projection inside the hole, and the outer periphery provides external clamp engagement.

[0032] In yet another embodiment, the present invention relates to a cooling plate configured for cooling a coolant and / or sample solution to a target temperature T2 which is between approximately 0°C and approximately the dew point temperature, preferably between approximately 0°C and 5°C, and more preferably between approximately 0°C and approximately 3°C.

[0033] Preferably, the cooling plate includes one or more (cooled) well plates for receiving a coolant and / or sample solution, one or more cooling elements configured to be positioned below the well plates, and preferably a metal block configured to be positioned below one or more cooling elements. In this way, great flexibility in use can be provided.

[0034] Preferably, one or more cooling elements are in contact with one or more (cooled) well plates and their cold sides, and the one or more cooling elements are preferably provided in the form of Peltier elements. This makes it possible to achieve optimal heat transfer characteristics.

[0035]

[0035] The present invention also relates to a system for carrying out the above method, comprising a pipette holding and cooling unit and a cooling plate as described above, and preferably a cryo-grid having a carbon layer and an additional gold layer.

[0036] A syringe pump according to the present invention is described in more detail below by exemplary embodiments and with reference to the accompanying drawings. [Brief explanation of the drawing]

[0037] [Figure 1] This shows a pipette holding and cooling unit according to the present invention. [Figure 2] A cooling plate according to the present invention is shown. [Figure 3] Microscopic images of a warm sample written on a cryo-grid are shown. [Figure 4] Microscopic images of a cold sample written on a cryo-grid are shown. [Modes for carrying out the invention]

[0038] In the following description, certain terms are used for convenience and are not intended to limit the invention. The terms “right,” “left,” “up,” “down,” “under,” and “above” refer to directions in the figures. Terminology includes the terms explicitly mentioned, as well as their derivatives and terms having similar meanings. Spatially relative terms (e.g., “beneath,” “below,” “lower,” “above,” “upper,” “proximal,” “distal,” etc.) may also be used to describe the relationship between one element or feature and another, as shown in the drawings. These spatially relative terms are intended to encompass different locations and directions of the device being used or operated, in addition to the locations and directions shown in the drawings. For example, if a device in a drawing is inverted, an element described as "below" or "beneath" other elements or features becomes "above" or "over" those elements or features. Therefore, the exemplary term "below" can encompass both above and below position and direction. Unless otherwise indicated, the device may be oriented in other directions (90-degree rotation or other directions), and the spatially relative descriptions used herein are interpreted accordingly. Similarly, descriptions of movement along various axes, and movement about various axes, include the position and direction for various specific devices.

[0039] To avoid repetition in the drawings and descriptions of various aspects and explanatory embodiments, it should be understood that many features are common to many aspects and embodiments. The omission of an aspect from the description or drawings does not mean that that aspect is missing from the embodiments in which it is incorporated. Rather, aspects may be omitted for clarity and to avoid redundant descriptions. In this context, the following applies to the remainder of this specification: to clarify the drawings, if a drawing contains reference numerals not described in the directly relevant part of the specification, a previous or subsequent explanatory section is referenced. Furthermore, for lucidity, if not all features of a component are referenced in a drawing, another drawing showing the same component is referenced. Similar numerals in two or more drawings represent identical or similar elements.

[0040] Figure 1 shows a pipette holding and cooling unit 1 according to the present invention. In this exemplary embodiment, the pipette holding and cooling unit 1 comprises a body 2 that is rotationally symmetric with respect to a T-shaped cross-section, and the body is formed from a metallic material (preferably brass or copper). The body 2 consists of a cylindrical plate 2.2 and a cylindrical extension 2.1 positioned perpendicular to the cylindrical plate 2.2. A coaxial clamp portion 4 is positioned at the distal end of the cylindrical extension portion 2.1 together with a capillary pipette 5. The cylindrical plate 2.2 and the cylindrical extension portion 2.1 of the body 2 include a passage 9 for the capillary pipette 5. The clamp portion 4 consists of an outer circumference 4.1 and a flexible, slightly conical insertion projection 4.2 (the insertion projection is positioned at the center inside the clamp portion 4). An insertion projection 4.2 is inserted into the distal end of the cylindrical extension 2.1 of the main body 2, and the outer circumference 4.1 is tightly screwed in to achieve a clamping fit for securing the capillary pipette 5. A cooling unit 3 is positioned below the cylindrical plate 2.2 of the main body 2, and the cooling unit includes a cooling element 3.1 (preferably a Peltier element) and a cooling block 3.2, the cooling block preferably being made of copper. A coolant (e.g., water) usually flows through the cooling block 3.2. The cooling element 3.1 is in a heat transfer relationship with the cylindrical plate 2.2 and the cooling block 3.2. To cool the cylindrical extension 2.1 of the main body 2, the cold side of the cooling element 3.1 or Peltier element is placed relative to the cylindrical plate 2.2 such that the area of ​​the pipette 5 receiving the sample solution reaches a desired target temperature T1 after sample aspiration. The water flowing through the cooling block 3.2 dissipates the heat generated by the cooling element 3.1 or the Peltier element.

[0041]

[0040] Figure 2 shows a cooling plate 6 according to the present invention. The cooling plate 6 of this exemplary embodiment comprises four cooled (well) plates 6.1, 6.2, 6.3 and 6.4. However, the cooling plate 6 may comprise any other number of plates, e.g., two, three, five, six, seven or eight cooled (well) plates, depending on the actual application. Generally, it is desirable that the cooling plate 6 provide a flexible cooling regime for both the coolant and the sample solution. In this embodiment, cooled (well) plates 6.1 and 6.2 are provided with blind-hole-shaped holes 6.1.1 and 6.2.1 for cooling the coolant, and cooled (well) plates 6.3 and 6.4 are provided with square recesses 6.3.1 and 6.4.1 for cooling the sample solution. However, the geometry of the well plates may vary depending on the actual purpose. Below the well plates 6.1, 6.2, 6.3, and 6.4, four independent cooling elements 7 (preferably Peltier elements) are arranged. The cold side of the cooling elements 7 and / or Peltier elements is in contact with the bottom surface of the cooled (well) plates 6.1, 6.2, 6.3, and 6.4. Thus, one Peltier element for each individual cooled (well) plate helps to further fine-tune the cooling process. Below the cooling elements 7 and / or Peltier elements 7, a metal block 8 (preferably made of copper) is arranged, which is configured to receive heat from the other side of the Peltier elements 7. The metal block 8 may also contain water. The cooling plates 6 of the present invention can efficiently and accurately cool the sample solution and coolant to the target temperature T2.

[0042] To better illustrate the concept of the present invention, Figure 3 is shown first. It is an overview of three exemplary microscopic grid images of “warm” samples written on different cryo-grids (before being inserted into liquid ethane and stored in grid boxes B1-B4, maintained at -195°C). Sample solutions were supplied from four different sources E1-E4. For each grid, the glow discharge was 900.0 volts for 60 seconds. Writing was performed at a flow rate of 0.25 nanoliters per second in each case, achieved by the associated pico-pump, but the time to prepare (i.e., “write") the sample on the cryo-grid varied between 4 and 5.7 seconds depending on the writing pattern. The writing speed of the preparations was 2 mm / s each. The sample layer thickness was between 1.0 μm and 1.2 μm. Path separation was between 0.11 mm and 0.14 mm. The total diameter of the samples was between 1.12 mm and 1.21 mm. The time delay after writing was 0 seconds in each case. The offset (i.e., the temperature difference between the dew point and the grid temperature) was between 0.20°C and 0.57°C. The warm samples showed significant thickness variation and numerous open holes within the grid square as a result of excessive evaporation.

[0043] Figure 4 then shows exemplary microscopic images of “cold” samples written on different cryo-grids, again to better illustrate the concept of the present invention (before insertion into liquid ethane and storage at positions B1 to B4). Here again, the sample solution was supplied from four different sources E1 to E4. The discharge was 1000.0 volts for 60 seconds. Writing was performed at flow rates of 0.1 nanoliters / second to 0.25 nanoliters / second and achieved by the associated pico-pump, but the time to prepare (i.e., “write") the cold sample on the cryo-grid varied between 4.6 seconds and 11 seconds, depending on the writing pattern and writing speeds of 1.0 mm / s and 2.5 mm / s. The layer thickness of grid E1 was 1.0 μm. Path separation was 0.11 mm in each case. The total diameter of the sample was 1.21 mm in each case. The time delay after cold writing was 0 seconds in each case. The offset (i.e., the temperature difference between the dew point and the grid temperature) was between -0.13°C and 0.03°C. The cold sample showed a homogeneous layer and numerous closed pores in the grid squares as shown in the exemplary microscopic overview grid image. The grid image with respect to source E2 is important as it shows liposomes, which are known to be highly sensitive to excessive evaporation.

[0044] From several experiments conducted by the applicant, it can be generally concluded that so-called "cold writing" leads to better sample preparation results (as shown in Figures 3 and 4). This is based on the following assumption: (i) Due to the precise pico pump, relatively thin sample layers, generally having a thickness between 1 μm and 3 μm, can be "written" onto the cryo grid; (ii) Depending on the offset (i.e., the temperature difference between the grid temperature and the dew point), the typical writing time is about 4 seconds to about 20 seconds (maximum), and it has been observed that a sample layer of 0.1 μm to 3 μm evaporates. This means that with a carbon film thickness of 12 nm, the sample layer may be too thin (i.e., less than 12 nm) and may not spread into a hole with a diameter of 1 μm; (iii) The cryo-grid was therefore cooled to the dew point or a temperature close to it (i.e., an offset of about 0°C) so that theoretically no more water would evaporate from the sample; (iv) Nevertheless, it was found that many grids, especially those with an optimally thin layer for microscopy, tend to dry out; (v) Thus, it was assumed that although the sample solution deposited on the cryo-grid could remove heat due to the dew point temperature of the copper bars of the grid, it would not cool sufficiently quickly. However, even with a sample layer thickness of 1 μm to 3 μm, the grid squares have sides of approximately 80 μm and are equipped with a carbon layer with holes of 1 μm in diameter. The 12 nm thick carbon layer does not contain sufficient heat capacity to cool the sample layer (it cools sufficiently quickly). Therefore, the concept of the present invention for cooling the pipette and sample solution (and coolant) in the manner described above has been demonstrated with compelling results.

[0045] This disclosure also individually covers all further features shown in the drawings, even if not described prior to or below. Furthermore, the drawings and specification, and one alternative example of such features, may be disclaimed from the subject matter of the invention or the disclosed subject matter. This disclosure includes subject matter consisting of features defined in the claims or exemplary embodiments, and subject matter containing such features.

[0046]

[0045] Furthermore, in the claims, the word “comprising” does not exclude other elements or processes, and the indefinite article “a” or “an” does not exclude plural. One unit or process may fulfill the function of several features mentioned in the claims. The fact that certain means are mentioned in mutually different dependent claims does not mean that combinations of these means cannot be used advantageously. Terms such as “essentially,” “about,” and “approximately” in relation to attributes or values ​​define the attribute or value precisely, particularly in relation to the attribute or a specific value. No reference numeral in the claims should be construed as limiting the scope. In the context of a given number or range, the term “about” refers, for example, to a value or range that is within 20%, 10%, 5%, or 2% of a given value or range. Components described as being coupled or connected may be directly coupled electrically or mechanically, or indirectly coupled through one or more intermediate components. [Explanation of Symbols]

[0047] 1: Pipette holding and cooling unit 2: Main body (preferably rotationally symmetric with respect to a T-shaped cross-section) 2.1: Cylindrical stretched section 2.2: Cylindrical Plate 2.3: Holes (preferably concentric and located at the distal end of the cylindrical extension) 3: Cooling unit 3.1: Cooling element (preferably a Peltier element) 3.2: Cooling Block 4: Clamp section (preferably coaxial) 4.1: Peripheral Sections 4.2: Insertion projection (preferably flexible and slightly conical) 5: Pipette (preferably a capillary pipette) 6: Cooling plate 6.1: Well plate (for coolant) 6.1.1: Blind hole-like hole 6.2: Well plate (for coolant) 6.2.1: Blind hole-like hole 6.3: Well plate (for sample solution) 6.3.1: Square recess 6.4: Well plate (for sample solution) 6.4.1: Square recess 7: Cooling element 8: Metal cooling block 9: Central channel for capillary pipette t d : Delay time between writing and insertion into liquid ethane T1: Target temperature (pipette) T2: Target temperature (sample / coolant)

Claims

1. A method for cooling a sample solution in a pipette configured for preparing a sample for electron microscopy, comprising the following steps: (a) The pipette (5) positioned within the pipette holding and cooling unit (1) is brought to a target temperature (T) in the range of approximately -2°C to approximately the dew point temperature. １ A cooling process; (b) Coolant and / or sample solution to a target temperature (T) in the range of approximately 0°C to approximately the dew point temperature. ２ A cooling process; (c) Step of aspirating the coolant using a pipette (5); (d) Step of aspirating the sample solution with a pipette (5); (e) A step of preparing a sample for electron microscopy on a sample support structure, wherein the sample support structure is preferably cooled to approximately the dew point temperature. Methods that include...

2. Target temperature (T １ The method according to claim 1, wherein the temperature is between approximately -1°C and approximately the dew point temperature, preferably between approximately 0°C and approximately the dew point temperature.

3. Target temperature (T ２ The method according to claim 1 or 2, wherein the temperature is between approximately 0°C and approximately 5°C, preferably between approximately 0°C and approximately 3°C.

4. The method according to any one of claims 1 to 3, wherein the sample in step e) is prepared on the sample support structure to a thickness of approximately 500 nm to approximately 3000 nm, preferably approximately 1000 nm to approximately 2000 nm, and more preferably approximately 1250 nm to approximately 1750 nm.

5. The method according to any one of claims 1 to 4, wherein the sample support structure is a cryo-grid.

6. The method according to claim 5, wherein the cryo-grid comprises a carbon layer and a gold layer.

7. After step (e), before the sample on the support structure is vitrified, a predetermined delay time (t ｄ The method according to claim 1, wherein the following is maintained.

8. A pipette holding and cooling unit (1) for providing cooling to a pipette (5), wherein the pipette holding and cooling unit (1) A pipette (5) configured for preparing samples for electron microscopy. A main body (2) configured to receive a pipette (5) inside, Cooling unit (3) for providing cooling to the main unit (2) The cooling unit (3) provides a cooling mechanism that cools the pipette (5) received inside the main body (2) to a target temperature (T) ranging from approximately -2°C to approximately the dew point temperature. １ A pipette holding and cooling unit (1) configured to cool to ).

9. Target temperature (T １ The pipette holding and cooling unit (1) according to claim 8, wherein the temperature is between approximately -1°C and approximately the dew point temperature, preferably between approximately 0°C and approximately the dew point temperature.

10. The pipette holding and cooling unit (1) according to claim 8 or 9, wherein the cooling unit (3) comprises a cooling element (3.1) and a cooling block (3.2), and the cooling element (3.1) is in a heat transfer relationship with the main body (2) and the cooling block (3.2).

11. The pipette holding and cooling unit (1) according to claim 10, wherein the cold side of the cooling element (3.1) is in contact with the main body (2), and the warm side of the cooling element (3.1) is in contact with the cooling block (3.2) to dissipate the heat generated by the cooling element (3.1).

12. The pipette holding and cooling unit (1) according to claim 10 or 11, wherein the cooling element (3.1) is in the form of a Peltier element.

13. The pipette holding and cooling unit (1) according to any one of claims 10 to 12, wherein the cooling block (3.2) is a metal block, preferably a metal block through which a coolant flows.

14. The pipette holding and cooling unit (1) according to any one of claims 8 to 13, wherein the pipette (5) is configured to prepare a sample on a sample support structure having a thickness of approximately 500 nm to approximately 3000 nm, preferably approximately 1000 nm to approximately 2000 nm, and more preferably approximately 1250 nm to approximately 1750 nm.

15. A pipette holding and cooling unit (1) according to any one of claims 8 to 14, comprising a clamp portion (4) for holding a pipette (5), wherein the clamp portion (4) is configured to be detachably attached to the main body (2).

16. The pipette holding and cooling unit (1) according to claim 15, wherein the main body (2) includes holes (2,3) configured to receive a clamp portion (4).

17. The coolant and / or sample solution should be at a target temperature (T) in the range between approximately 0°C and approximately the dew point temperature. ２ A cooling plate (6) configured to cool the area.

18. A cooling plate (6) according to claim 17, comprising one or more well plates (6.1, 6.2; 6.3, 6.4) for receiving a coolant and / or sample solution, one or more cooling elements (7) configured to be positioned below the well plates (6.1, 6.2; 6.3, 6.4), and preferably a metal block (8) configured to be positioned below the one or more cooling elements (7).

19. The cooling plate (6) according to claim 18, wherein one or more cooling elements (7) are in contact with one or more well plates (6.1, 6.2; 6.3, 6.4) on their cold side, and the one or more cooling elements are preferably provided in the form of Peltier elements.

20. A system for performing the method according to any one of claims 1 to 7, comprising a pipette holding and cooling unit (1) according to any one of claims 8 to 16 and a cooling plate according to any one of claims 17 to 19.

Images