Method for cooling a sample solution in a pipette, a pipette holding and cooling unit and a 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) cooling a pipette (5) arranged within a pipette holding unit (1) to a target temperature (T1) in a range of approximately -2 °C to approximately dew point temperature; (b) cooling a cooling liquid and / or a sample solution to a target temperature (T2) in the range of approximately 0 °C to approximately dew point temperature; (c) aspirating cooling liquid with the pipette (5); (d) aspirating sample solution with the pipette (5); and (e) preparing a sample for electron microscopy on a sample support structure which is preferably cooled to approximately dew point temperature. The present invention also relates to a pipette holding and cooling unit (1) and a cooling plate (6).

Description

DESCRIPTIONTitleMETHOD FOR COOLING A SAMPLE SOLUTION IN A PIPETTE, A PIPETTE HOLDING AND COOLING UNIT AND A COOLING PLATETechnical Field

[0001] The present invention relates to a method for cooling a sample solution in a pipette configured for preparing a sample for electron microscopy, a pipette holding and cooling unit for cooling such a pipette, a cooling unit for cooling a cooling liquid and / or a sample solution and a system for carrying out the method.Background Art

[0002] As a novel method for preparing samples for electron microscopy, cryo-writing is increasingly gaining importance. Cryo-writing is particularly used for preparing respectively writing cryo grids for cryo electron microscopy with e.g. an aqueous protein sample solution by means of a respective pipette. Thereby, the pipette used for cryo- writing aspirates the aqueous protein solution from an appropriate container to release the latter when writing a pattern on the grid. Such technique is subject of WO 2017 / 005297 A1.

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

[0004] The layer thickness is crucial to be able to determine the atomic structure of a protein or protein complex. If the layer is too thin, the protein will denature at the air-water interfaces of the aqueous layer. If the layer is too thick, the protein will not produce sufficient image contrast when irradiated with the electron beam compared to the thicksurrounding aqueous layer. Producing layers with the right thickness is therefore a primary goal of all sample vitrification protocols for cryo electron microscopy.

[0005] The pump device used for cryo-writing (e.g. a pico-pump as described in applicant’s not yet published Swiss patent application CH 000225 / 2023) and the pm accuracy of the pipette movements are important prerequisites for the reproducible writing of sample layers of a selected thickness.

[0006] Controlling sample evaporation is also an important issue. In a 100% relative humidity environment, evaporation is negligible; it is suppressed at high relative humidity and / or when the sample layer is near the dew point temperature. The latter is the subject of WO 2018 / 073242 A1.

[0007] While the grid itself can be cooled by the grid holder (e.g. a gripper as described in applicant’s not yet published Swiss patent application CH 001089 / 2022), the heat of the applied sample solution must be transported to the heat sink represented by the grid structure.

[0008] If one considers the grid geometry with its 10 to 20 nm thick carbon layer spanning the grid squares of typically 80 pm side length, it becomes clear that a usually 100 times thicker sample layer does not reach the dew point before significant evaporation has taken place. If, on the other hand, the sample solution and the grid are kept at dew point, no evaporation will occur, and the vitrified layer could be too thick for (cryo-) electron microscopy.

[0009] Many proteins are not stable when extracted from their natural environment. Membrane proteins are particularly prone to denaturation when extracted from the lipid bilayer by detergents. But the solubilization is the prerequisite for the purification and cryo electron microscopy of membrane proteins. To keep such solutions cold helps stabilizing the membrane proteins.

[0010] It is therefore the object of the present invention to provide for a cooling method for the protein sample solution and the pipette by means of which an appropriate sample layer thickness may be ensured in a reproducible manner; also a respective pipette holding and cooling unit and a respective cooling plate shall be provided.Disclosure of the Invention

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

[0012] In one aspect, the 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) cooling a pipette arranged within a pipette holding and cooling unit to a target temperature Ti in a range of approximately -2 °C to approximately dew point temperature; (b) cooling a cooling liquid and / or a sample solution to a target temperature T2 in a range of approximately 0 °C to approximately dew point temperature; (c) aspirating cooling liquid with the pipette; (d) aspirating sample solution with the pipette; and (e) preparing (“writing”) a sample for electron microscopy on a sample support structure which is preferably cooled to approximately dew point temperature.

[0013] The advantage of the inventive method is to have a precise control of the deposit sample solution temperature, i.e. in connection with the sample support structure which is usually cooled to about dew point temperature by the gripper (as is the case in the aforementioned gripper application of applicant) and / or by the clamp holding the grid for writing, which allows the layer thickness to be tuned by the temperature offset of the grid temperature from dew point temperature and the time delay td between writing and insertion into e.g. liquid ethane. Thereby, the temperature offset should generally not be too large for a sufficient precise layer thickness control. However, without the appropriate cooling of the pipette and the sample solution (and the cooling liquid), the deposit sample might nevertheless evaporate too fast. Thus, only with this “cold writing” method sample layers of reproducible pre-determined thickness are reliably created and fragile proteins and their complexes are reliably stabilized.

[0014] It will be understood that generally in an atmosphere with a relatively low humidity and a relatively low dew point (e.g. in winter time), a comparatively high evaporation of the deposit sample solution takes place such that the protein may denaturate, whereas in an atmosphere with a relatively high humidity and a relatively high dew point (e.g. in summer time), a comparatively low evaporation of the deposit samplesolution takes place such that the layer thickness of the deposit sample solutions may remain too thick in order to ensure a sufficient resolution under the microscope.

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

[0016] The term “pipette” as used herein, particularly relates to micropipettes produced from glass capillaries which are combined with a pico-pump for dosing of liquids. The pipette may be used in connection with a pipette holding unit, which allows the pipette to be cooled.

[0017] The term “electron microscopy” as used herein, particularly relates to cryoelectron microscopy (cryo-EM).

[0018] The “dew point temperature” is the temperature air needs to be cooled to (at constant pressure) in order to achieve a relative humidity (RH) of 100%. At this point, the air cannot hold more water in the gas form. The dew point is affected by the air's humidity and pressure. The more moisture the air contains, the higher its dew point.

[0019] The “sample support structure” as used herein is usually in the form of cryo-grids respectively cryo-EM-grids which regularly comprise a mesh-like geometry coated with a holey carbon film having a thickness of 10 nm to 20 nm.

[0020] Preferably, the target temperature Ti for the pipette arranged within the pipette holding unit is between approximately -1 °C and approximately dew point temperature, preferably between approximately 0 °C and approximately dew point temperature. These temperature ranges have proven to provide for the best results in particular for cryowriting purposes.

[0021] Preferably, the target temperature T2 for cooling the liquid and / or the sample solution is between approximately 0 °C and approximately 5 °C, preferably between approximately 0 °C and approximately 3 °C. In this manner, the pipette may be cooled from inside and the efficiency of the layer thickness tuning for the cryo-writing procedure can be further enhanced such that electron microscopy with very good resolution may be achieved.

[0022] Preferably, the pipette is configured to prepare samples 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 and approximately 1750 nm on a sample support structure. In this manner, optimal preconditions for the cooling and cryo-writing process may 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 respectively the desired deposited sample temperature.

[0024] Preferably, after step (e), a pre-defined delay time period td is maintained, before the sample on the support structure is vitrified. This delay after writing prior to vitrification (i.e., plunging the sample support structure respectively grid into liquid ethane) is also effective in tuning the layer thickness of the sample solution deposited on the sample support structure respectively grid. The optimal delay time period depends on the initial layer thickness and temperature of the deposit sample solution and on the temperature of the sample support structure respectively grid.

[0025] In a further aspect, the invention relates to a pipette holding and cooling unit for providing cooling of a pipette. The pipette holding and cooling unit comprises a pipette configured for preparing a sample for electron microscopy, a main body configured for receiving the pipette therein and a cooling unit for providing cooling of the main body. The cooling unit is configured to cool the pipette received in the main body to a target temperature Ti in a range of approximately -2 °C to approximately dew point temperature.

[0026] Preferably, the target temperature Ti is between approximately -1 °C and approximately dew point temperature, preferably between approximately 0 °C and approximately dew point temperature. These temperature ranges have proven to provide for the best results in particular for cryo-writing purposes.

[0027] Preferably, the cooling unit comprises a cooling element and a cooling block wherein the cooling element is in heat transfer relation with the main body and the cooling block. Particularly preferred, a cold side of the cooling element abuts the main body and a warm side of the cooling element abuts the cooling block for dissipation of the heatgenerated by the cooling element. In this manner, a particularly accurate and uniform cooling effect may 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 through which preferably a coolant flows (e.g. water). Hereby, ideal heat transfer characteristics may be accomplished.

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

[0030] Preferably, the pipette holding and cooling unit comprises a clamping part in which the pipette is held, the clamping part being configured for releasable mounting at the main body. By means of the clamping unit, an easy exchangeability of the pipette is provided without the need of any additional equipment.

[0031] Preferably, the main body of the pipette holding and cooling unit comprises a bore being configured for receiving the clamping part. The clamping part advantageously comprises a flexible circumferential section and a centrally arranged insertion projection. The insertion projection is introduced into the bore of the main body at its distal end and the circumferential section is pushed over the distal end portion of the main body such that the insertion projection is received within the bore and the circumferential section provides for a clamping engagement from outside.

[0032] In a still further aspect, the invention relates to a cooling plate configured for cooling a cooling liquid and / or a sample solution to a target temperature T2 of between approximately 0 °C and approximately 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 comprises one or more (cooled) well plates for receiving cooling liquid and / or sample solution, one or more cooling element(s) arranged below the well plates and preferably a metal block arranged below the one or more cooling element(s). In this manner, a great flexibility in use can be provided.

[0034] Preferably, the one or more cooling element(s) abut the one or more (cooled) well plates with their cold side and wherein the one or more cooling element(s) are preferably provided in the form of Peltier elements. Hereby, optimal heat transfer characteristics may be achieved.

[0035] The invention also relates to a system for carrying out the afore-described method which comprises a pipette holding and cooling unit and a cooling plate as outlined above as well as preferably a cryo-grid with a carbon layer and an additional gold layer.Brief Description of the Drawings

[0036] The syringe pump according to the present invention is described in more detail hereinbelow by way of an exemplary embodiment and with reference to the attached drawings, in which:Fig. 1 shows a pipette holding and cooling unit in accordance with the present invention;Fig. 2 shows a cooling plate in accordance with the present invention;Fig. 3 shows microscopic images of warm samples written on a cryo-grid; andFig. 4 shows microscopic images of cold samples written on a cryo-grid.Description of Embodiments

[0037] In the following description certain terms are used for reasons of convenience and are not intended to limit the invention. The terms “right”, “left”, “up”, “down”, “under" and “above" refer to directions in the figures. The terminology comprises the explicitly mentioned terms as well as their derivations and terms with a similar meaning. Also, spatially relative terms, such as "beneath", "below", "lower", "above", "upper", "proximal", "distal", and the like, may be used to describe one element's or feature's relationship to another element or feature as illustrated in the figures. These spatially relative terms are intended to encompass different positions and orientations of the devices in use or operation in addition to the position and orientation shown in the figures. For example, if a device in the figures is turned over, elements described as "below" or "beneath" other elements or features would then be "above" or "over" the other elements or features. Thus, the exemplary term "below" can encompass both positions and orientations of above and below. The devices may be otherwise oriented (rotated 90degrees or at other orientations), and the spatially relative descriptors used herein interpreted accordingly. Likewise, descriptions of movement along and around various axes include various special device positions and orientations.

[0038] To avoid repetition in the figures and the descriptions of the various aspects and illustrative embodiments, it should be understood that many features are common to many aspects and embodiments. Omission of an aspect from a description or figure does not imply that the aspect is missing from embodiments that incorporate that aspect. Instead, the aspect may have been omitted for clarity and to avoid prolix description. In this context, the following applies to the rest of this description: If, in order to clarify the drawings, a figure contains reference signs which are not explained in the directly associated part of the description, then it is referred to previous or following description sections. Further, for reason of lucidity, if in a drawing not all features of a part are provided with reference signs it is referred to other drawings showing the same part. Like numbers in two or more figures represent the same or similar elements.

[0039] Fig. 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 rotationally symmetrical base body 2 with a T-shaped cross section, which is formed from metal material (preferably brass or copper). The base body 2 consists of a cylindrical plate 2.2 and a cylindrical extension 2.1 aligned perpendicularly to the cylindrical plate 2.2. At the distal end of the cylindrical extension 2.1 , a coaxial clamping part 4 is arranged together with the capillary pipette 5. The cylindrical plate 2.2 and the cylindrical extension 2.1 of the base body 2 comprise a passage 9 for the capillary pipette 5. The clamping part 4 consists of a peripheral section 4.1 and a flexible, slightly conical insertion projection 4.2, which is arranged centrally within the clamping part 4. The insertion projection 4.2 is inserted into the distal end of the cylindrical extension 2.1 of the base body 2 and the peripheral section 4.1 is screwed tight in order to achieve a clamping fit for fastening the capillary pipette 5. Below the cylindrical plate 2.2 of the main body 2, a cooling unit 3 is arranged, which comprises a cooling element 3.1 (preferably a Peltier element) and a cooling block 3.2, which is preferably made of copper. A coolant (e.g., water) usually flows through the cooling block 3.2. The cooling element 3.1 is in heat transfer relationship with the cylindrical plate 2.2 and the cooling block 3.2. The cold side of the cooling element 3.1 or Peltier element rests against the cylindrical plate 2.2 in order to cool the cylindrical extension 2.1 of the base body 2, so that the area of the pipette 5in which the sample solution is received reaches the desired target temperature Ti after sample aspiration. The water flowing through the cooling block 3.2 dissipates the heat generated by the cooling element 3.1 or Peltier element.

[0040] Fig. 2 illustrates a cooling plate 6 in accordance with 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, the cooling plate 6 shall provide for a flexible cooling regime for both, cooling liquids and sample solutions. In the present embodiment the cooled (well) plates 6.1 and 6.2 with the blind-hole-shaped bores 6.1.1 and 6.2.1 are provided for the cooling of cooling liquid and the cooled (well) plates 6.3 and 6.4 with the square shaped recesses 6.3.1 and 6.4.1 are provided for the cooling of sample solution. Yet, the geometry of the well-plates may be different, depending on the actual purpose. Below the well plates 6.1 , 6.2, 6.3 and 6.4 there are arranged four independent cooling elements 7 (preferably Peltier elements). The cooling elements 7 respectively Peltier elements abut with their cold side the bottom side of the cooled (well) plates 6.1 , 6.2, 6.3 and 6.4. Thus, one Peltier element for each individual cooled (well) plate serves for further fine-tuning of the cooling process. Below the cooling elements 7 respectively Peltier elements there is arranged a metal block 8 (preferably made of copper) which is configured for receiving heat from the other side of the Peltier elements 7. The metal block 8 may also contain water. By means of the inventive cooling plate 6, the sample solutions and the cooling liquids may be cooled in an efficient and precise manner to the target temperature T2.

[0041] Fig. 3 initially shows, for better illustration of the inventive concept, three exemplary microscopic overview grid images of “warm” samples written onto different cryo-grids before insertion into liquid ethane and storage in positions B1 to B4 of a grid box kept at -195 °C. The sample solutions were provided from four different sources E1 to E4. For each grid the glow discharge was at 900.0 Volt for 60 seconds. Writing was with a flow rate of 0.25 nanoliter per second in each case and was achieved with the associated pico-pump, but the time for preparing (i.e. , “writing”) the sample on the cryo- grids varied between 4 seconds and 5.7 seconds depending on the writing pattern. The preparation respectively writing speed was 2 mm / s. The layer thickness of the sample was between 1.0 pm and 1.2 pm. The path separation was between 0.1 1 mm and 0.14 mm. The total diameter of the sample was between 1.12 mm and 1.21 mm. The timedelay 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 substantially showed strong thickness variations and many open holes within the grid squares as result of excessive evaporation.

[0042] Subsequently, Fig. 4 shows, also for better illustration of the inventive concept, exemplary microscopic images of “cold” samples written onto different cryo-grids before insertion into liquid ethane and storage in positions B1 to B4. The sample solutions were again provided from four different sources E1 to E4. The discharge was at 1000.0 Volt for 60 seconds. Writing was with a flow rate of between 0.1 nanoliter per second and 0.25 nanoliter per second and was achieved with the associated pico-pump, but the time for preparing (i.e., “writing”) the cold sample on the cryo-grids varied between 4.6 seconds and 11 seconds depending on the writing pattern and writing speed of 1 .0 mm / s and 2.5 mm / s. The layer thickness of grid E1 was 1 .0 pm. The path separation was 0.1 1 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 samples substantially showed homogenous layers and many closed holes in the grid squares displayed on the exemplary microscopic overview grid images. Importantly the image of grid relating to source E2 shows liposomes that are known to be highly sensitive to excessive evaporation.

[0043] From multiple experiments conducted by the applicant it may generally be concluded that so-called “cold writing” provides better sample preparation results (as demonstrated by Figs. 3 and 4). This is based on the following assumptions: (i) due the precise pico-pumps, relatively thin sample layers with thicknesses of generally between 1 pm and 3 pm may be “written” on the cryo-grids; (ii) depending on the offset (i.e. the temperature difference between the grid temperature and the dew point) it has been observed that 0.1 pm to 3 pm of the sample layers evaporate since the time for writing usually is about 4 seconds to about 20 seconds (max) which means that the sample layers might get too thin (i.e. smaller than 12 nm) to span a hole with diameter of 1 pm in a carbon film 12 nm thickness; (iii) hence, the cryo-grid was cooled to a temperature at or near the dew point (i.e. an offset of about 0 °C) such that, theoretically, no more water should evaporate from the sample; (iv) nevertheless, it was found that many grids, in particular with optimally thin layers for microscopy, tend to dry out; (v) hence, it wasassumed that the sample solution when deposited on the cryo-grid does not cool down fast enough since - although the copper bars of the grids are at dew point temperature and may thus remove heat. However, even if the sample layer is only 1 pm to 3 pm thick, the grid square comprises a side length of about 80 pm, and the carbon layer with its 1 pm diameter holes and its 12 nm thickness does not comprise sufficient heat capacity to cool the sample layer (fast enough). Therefore, the inventive concept of cooling the pipette and the sample solution (and the cooling liquid) in the above-described manner has been established with convincing results.

[0044] The present disclosure also covers all further features shown in the Figs, individually although they may not have been described in the afore or following description. Also, single alternatives of the embodiments described in the figures and the description and single alternatives of features thereof can be disclaimed from the subject matter of the invention or from disclosed subject matter. The disclosure comprises subject matter consisting of the features defined in the claims or the exemplary embodiments as well as subject matter comprising said features.

[0045] Furthermore, in the claims the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. A single unit or step may fulfil the functions of several features recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. The terms “essentially”, “about”, “approximately” and the like in connection with an attribute or a value particularly also define exactly the attribute or exactly the value, respectively. The term “about” in the context of a given numerate value or range refers to a value or range that is, e.g., within 20%, within 10%, within 5%, or within 2% of the given value or range. Components described as coupled or connected may be electrically or mechanically directly coupled, or they may be indirectly coupled via one or more intermediate components. Any reference signs in the claims should not be construed as limiting the scope.List of reference numbers:1 pipette holding and cooling unit2 main body (preferably rotationally symmetrical with a T-shaped cross section)2.1 cylindrical extension2.2 cylindrical plate2.3 bore (preferably concentric and arranged at the distal end of the cylindrical extension)3 cooling unit3.1 cooling element (preferably a Peltier element)3.2 cooling block4 clamping part (preferably coaxial)4.1 peripheral section4.2 insertion projection (preferably flexible and slightly conical)5 pipette (preferably a capillary pipette)6 cooling plate6.1 well plate (for cooling liquid)6.1.1 blind-hole-shaped bores6.2 well plate (for cooling liquid)6.2.1 blind-hole shaped bores6.3 well plate (for sample solution)6.3.1 square-shaped recesses6.4 well plate (for sample solution)6.4.1 square-shaped recess7 cooling elements8 metal cooling block9 central passage for capillary pipette td delay time period between writing and insertion into liquid ethaneTi target temperature (pipette)T2 target temperature (sample / cooling liquid)

Claims

CLAIMS1 . 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) cooling a pipette (5) arranged within a pipette holding and cooling unit (1 ) to a target temperature (Ti) in a range of approximately -2 °C to approximately dew point temperature;(b) cooling a cooling liquid and / or a sample solution to a target temperature (T2) in a range of approximately 0 °C to approximately dew point temperature;(c) aspirating cooling liquid with the pipette (5);(d) aspirating sample solution with the pipette (5);(e) preparing a sample for electron microscopy on a sample support structure which is preferably cooled to approximately dew point temperature.

2. The method according to claim 1 , wherein the target temperature (T1) is between approximately -1 °C and approximately dew point temperature, preferably between approximately 0 °C and approximately dew point temperature.

3. The method according to claim 1 or 2, wherein the target temperature (T2) 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 the preceding claims, wherein the samples in step (e) are prepared 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 and approximately 1750 nm on a sample support structure.

5. The method according to any one of the preceding claims, 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. The method according to claim 1 , wherein after step (e), a pre-defined delay time period (td) is maintained, before the sample on the support structure is vitrified.8 A pipette holding and cooling unit (1 ) for providing cooling of a pipette (5), the pipette holding unit (1 ) comprising a pipette (5) configured for preparing a sample for electron microscopy, a main body (2) configured for receiving the pipette (5) therein, a cooling unit (3) for providing cooling of the main body (2), wherein the cooling unit (3) is configured to cool the pipette (5) received in the main body (2) to a target temperature (Ti) in a range of approximately -2 °C to approximately dew point temperature.

9. The pipette holding and cooling unit (1 ) according to claim 8, wherein the target temperature (Ti) is between approximately -1 °C and approximately dew point temperature, preferably between approximately 0 °C and approximately 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) wherein the cooling element (3.1 ) is in heat transfer relation with the main body (2) and the cooling block (3.2).11 . The pipette holding and cooling unit (1 ) according to claim 10, wherein a cold side of the cooling element (3.1 ) abuts the main body (2), and a warm side of the cooling element (3.1 ) abuts the cooling block (3.2) for dissipation of the heat generated by the cooling element (3.1 ).

12. The pipette holding and cooling unit (1 ) according to any one of claims 10 to 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 through which preferably 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 samples with a thickness of approximately 500 nm to approximately 3000 nm, preferably about 1000 nm to about 2000 nm and more preferably approximately 1250 nm and approximately 1750 nm on a sample support structure.

15. The pipette holding and cooling unit (1 ) according to any one claims 8 to 14, comprising a clamping part (4) in which the pipette (5) is held, the clamping part (4) being configured for releasable mounting at the main body (2).

16. The pipette holding and cooling unit (1 ) according to claim 15, wherein the main body (2) comprises a bore (2.3) being configured for receiving the clamping part (4).

17. A cooling plate (6) configured for cooling a cooling liquid and / or a sample solution to a target temperature (T2) in a range between approximately 0 °C and approximately dew point temperature.

18. The cooling plate (6) according to claim 17, comprising one or more well plates (6.1 , 6.2; 6.3, 6.4) for receiving cooling liquid and / or sample solution, one or more cooling element(s) (7) arranged below the well plates (6.1 , 6.2; 6.3, 6.4) and preferably a metal block (8) arranged below the one or more cooling element(s) (7).

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

20. A system for carrying out 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.

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