A microfluidic reactor device for use in a cryo-electron microscope, a mounting stage for such microfluidic reactor device and a method of analysing a sample under cryogen temperature conditions in a cryo-electron microscope.

Abstract

A microfluidic reactor device for use in a cryo-electron microscope is proposed. The microfluidic reactor device comprises at least a first reactor layer being at least partly transparent to an electron beam of the cryo-electron microscope. The first reactor layer exhibits a first surface layer side and a second surface layer side opposite from the first surface layer side, wherein at least part of the first surface layer side is structured - during use - to function as a reactor surface for accommodating a sample to be analysed. The second surface layer side is structured - during use - to be brought in thermal contact with a cryogen. In addition, heating means are provided for heating at least the reactor surface of the first surface layer side. Moreover, a second reactor layer being at least partly transparent to the electron beam of the cryo-electron microscope is provided, the second reactor layer facing and extending next to the first reactor layer at a mutual distance from each other, thereby forming a rector chamber between the first and the second reactor layers.

Description

[0001] TITLE

[0002] A microfluidic reactor device for use in a cryo-electron microscope, a mounting stage for such microfluidic reactor device and a method of analysing a sample under cryogen temperature conditions in a cryo-electron microscope.

[0003] TECHNICAL FIELD

[0004] The present disclosure relates to the field of technology of implementing cryo-electron microscopes for studying samples or specimens with a high resolution, for instance with a resolution higher than 0.2 nm, more in particular higher than 0.12 nm. To this end, a Micro Electro Mechanical System (MEMS)-based sample carrier device containing a sample or specimen to be analysed is placed in an electron beam of an electron microscope, while an image generated by the beam is displayed on, for instance, a fluorescence screen or is captured with the aid of a camera. Studying these specimens is usually done in a vacuum and at room temperature.

[0005] BACKGROUND OF THE DISCLOSURE

[0006] An example of a microfluidic reactor device for use in an electron microscope is for example disclosed in the International patent application no. W02006 / 031104A1. However, this known example is unsuited for implementation ins so-called cryo-electron microscopy (cryo-EM) analysis. Cryo-electron microscopy requires vitrification (i.e. the formation of amorphous ice) of biological samples on an analysis surface. In known techniques, the vitrification is achieved by freezing the sample to be analysed at an extremely fast cooling (freezing) rate. There are nowadays different techniques to achieve fast cooling (freezing) rates, including plunge freezing and jetting. However, the vitrification remains as the main bottleneck for cryo-EM suffering from extremely poor reproducibility, it is very time consuming, the results are user-dependent and the air-water interface negatively affects the sample integrity.

[0007] Since the introduction of cryo-EM, the sample preparation, the sample carrier, and vitrification method have remained unchanged. All the vitrification methods require the use of specifically designed surface platforms and a cryogen. After sample deposition (typically drop-casted) on the analysis surface, the latter is either plunged in the cryogen or the cryogen is sprayed / jetted on the analysis surface to achieve vitreous ice. Current vitrification machines that use such analysis surfaces are extremely costly and complex with a large footprint.

[0008] Accordingly, it is a goal of the present disclosure to provide an improved and smart sample carrier device, which is MEMS-based, that can be used in an efficient and reproducible manner in a cryo-electron microscope application, having a less complex design and that reduces the size and the price of the vitrification machine.

[0009] SUMMARY OF THE DISCLOSURE

[0010] According to a first example of the disclosure, a microfluidic reactor device for use in a cryo-electron microscope is proposed. In a first generic example the microfluidic reactor device can be configured as a sample carrier embodied as a MEMS-based device that comprises at least a first reactor layer being at least partly transparent to an electron beam of the cryo-electron microscope. The first reactor layer exhibits a first surface layer side and a second surface layer side opposite from the first surface layer side, wherein at least part of the first surface layer side is structured - during use - to function as a reactor surface for accommodating a sample to be analysed. The second surface layer side is structured - during use - to be brought in thermal contact with a cryogen.

[0011] In addition, heating means are provided for heating at least the reactor surface of the first surface layer side. Moreover, as an optional feature, at least one pair of electrodes are implemented that are structured - during use - to monitor the characteristics of the sample accommodated on the reactor surface.

[0012] This compact design of the microfluidic reactor or smart sample carrier device will allow easy, fast, automated and reproducible on-chip vitrification of samples for cryo-EM applications upon sample deposition (e.g. via the conventional drop-casting or any other sample deposition method). This will result in a smarter and much more robust alternative to the conventional cryo-EM sample carriers (i.e. the 3 mm metallic grids). In addition, it will enable users to explore new techniques such as time-resolved cryo-EM, as the user can define the exact moment in time when the on-chip vitrification should be triggered. Furthermore, the optional configuration with the integrated nano-sensors on the MEMS device enables the user to immediately monitor various material characteristics, such as cryogenic characteristics e.g. the ice quality of the vitrified sample (e.g. thickness of the ice, crystallinity, etc). This will prevent users from proceeding with a faulty batch and wasting valuable transmission electron microscopy (TEM) time.

[0013] Apart from monitoring and detecting certain cryogenic characteristics, the at least one pair of electrodes can also be used to fine-tune the wettability (contact angle) of the surface via electrowetting. Moreover, with the application of an electric field via the at least one pair of electrodes, any proteins (or any other type of biomolecule) in the sample can be manipulated during the sample deposition on the reactor surface. This manipulation allows for electrophoresis to separate the particles (e.g., by weight / size), to prevent particle agglomeration, or to rotate the proteins in the sample and align them with the electric field being applied between the electrodes.

[0014] In a particular advantageous example, the at least one pair of electrodes are mounted in or on the first reactor layer, optionally at least partially encapsulated in the first reactor layer, or more optionally completely encapsulated in the first reactor layer. This ensures an effective measurement contact between the electrode pair and the sample to be analysed as being present directly above the pair of electrodes on the reactor surface of the first reactor layer.

[0015] More in importantly, the at least one pair of electrodes may extend across the reactor surface of the first reactor layer thus ensuring improved detection and a more accurate monitoring and measurement of the various material (cryogenic) characteristics over a sufficiently large contact area.

[0016] In a first example of the pair of electrodes, these electrodes are each configured as single segment electrodes positioned at a certain distance from each other. This allows for a fast position dependent measurement technique. In a more sophisticated example, allowing accurate monitoring and measurement of the various material (cryogenic) characteristics over a sufficiently large contact area, the at least one pair of electrodes are each configured as multi-segment electrodes, which multi-segment electrodes intermesh with each other. In a further advantageous example, the at least one pair of multi-segment electrodes are concentrically positioned with respect to each other.

[0017] In particular, the at least one pair of electrodes are made of metal (e.g. Au or Pt). Optionally, the electrodes may be provided with an adhesion layer made of Ta, Cr or Ti, which improves adhesion to the first reactor layer, in particular in the example that the first reactor layer is made from of SiNx. Furthermore, the at least one pair of electrodes can also be made of glassy carbon, as this is a highly conductive and chemically inert material.

[0018] In the example of fully encapsulated electrodes, the pair of electrodes may be made of Molybdenum (Mo) similar as the material of which the heating means (heating element or heating coil) is made of. That will simplify the fabrication.

[0019] A typical non-limiting thickness of the electrodes ranges between 50 nm and 250 nm, with a length of approx. 20 pm long and a width of approx. 10 pm, with a spacing of 1 m distance between the pair of electrodes.

[0020] In a further advantageous example, the microfluidic reactor device or MEMS- based device according to the disclosure could be further provided with a second reactor layer that is at least partly transparent to the electron beam of the cryo-electron microscope. The second reactor layer is facing and extending next to the first reactor layer at a mutual distance from each other and between which a microfluidic reactor chamber is formed. Additionally, an inlet and an outlet are provided for feeding the sample (suspended in its liquid native environment) through the reactor chamber and across the reactor surface.

[0021] This example of the resulting microfluidic reactor device according to the disclosure provides additional advantages. Instead of being limited to exclusively dropcasting the sample, the user can flow the sample into the resulting microfluidic reactor while it is in its native liquid environment. The presence of a closed nano-channel or microfluidic reactor chamber formed between the reactor layers eliminates any air-water interface problems during vitrification analysis.

[0022] The inlet and outlet may be considered to be openings between the first and second reactor layer provided along the entire periphery of the microfluidic reactor device or MEMS-based device, wherein the inlet and the outlet overlap substantially.

[0023] In particular the inlet and the outlet are provided in and through the first reactor layer.

[0024] Preferably, at least the parts of the first and / or second reactor layers which are transparent to the electron beam are at a mutual distance of on average less than 100 nanometres, more in particular less than 20 nanometres and preferably less than 10 nanometres.

[0025] In particular, the mutual distance between the first reactor layer and the second reactor layer can also be a few micrometres (to enable cell culturing and full cell vitrification), wherein the reactor chamber enclosed between the parts of the reactor layers has an exposed surface, measured approximately parallel to the parts of the reactor layers, which is less than 20 mm2, in particular less than 10 mm2, more in particular less than 5 mm2and preferably of the order of magnitude of 1 mm2.

[0026] In a preferred example, the first and second reactor layers are gas tight and liquid tight, thus preventing any leakage and / or contamination from outside the microfluidic reactor device into the reactor chamber.

[0027] In further advantageous example according to the disclosure windows are provided in the first and second reactor layers, which windows are transparent to the electron beam. Preferably at least one window in the first reactor layer is located opposite to a window in the opposite second reactor layer. In particular the windows present in the respective reactor layer is more transparent to the electron beam than the remaining part of the respective reactor layer. To this end, for instance, the reactor layer can have been thinned locally, thereby forming a recess whose bottom part is formed by the window, and which has an increased transparency to the electron beam. In another example the reactor layer can be provided with a complete through hole, which is subsequently covered by a graphene layer.

[0028] Here, each window has a surface which is particularly small compared with the total reactor surface of the reactor chamber, for instance a few square micrometres. Sample to be analysed on a micrometre or even nanometre scale can be provided on such a window part of the reactor surface so that a still better image can be obtained.

[0029] In particular, the heating means comprise a heating element, for example but not limiting a heater coil, which heating element is received in or on the first reactor layer. Preferably, a heating element according to the disclosure is manufactured from molybdenum (Mo) like the encapsulated pair of electrodes or titanium nitrate. In an example, for instance in the form of strips and / or plates with a particularly small thickness, for instance less than 500 nanometres, more in particular for instance 300 nanometres or less. Thus, the heating element can particularly simply be included in the first reactor layer or be formed therein, without the reactor chamber being undesirably influenced with regard to shape and dimensions.

[0030] In a further advantageous example, the microfluidic reactor device is formed as a monolithic component, which eliminates the need of having to assemble a top and a bottom layer. This monolithic component ensures complete consistency in layer thickness from batch to batch.

[0031] The present disclosure also pertains to a mounting stage which stage is designed to mount the microfluidic reactor or MEMS-based device (embodied as either the bottom chip only for on-chip vitrification of drop-casted samples, or as a microfluidic reactor, both configured as a dual-cell chip or as a monolithic device both incorporating a microfluidic reactor chamber, so that the user flows in the sample) according to the disclosure. The mounting stage is formed of at least a first stage component for accommodating the second reactor layer of the microfluidic reactor device (thus configured as a MEMS-based device), and structured to be brought in thermal contact with a cryogen, as well as a control unit for controlling the heating means

[0032] In an example of the mounting stage the control unit is structured for detecting a resistance or capacitance between the at least one pair of electrodes and for determining one or more material (cryogenic) characteristics of the sample accommodated on the reactor surface based on the resistance or capacitance thus detected.

[0033] Optionally the first stage component can be provided with a recess for accommodating the first reactor layer of the microfluidic reactor device I M EMS- based device. By designing the recess of having inner dimension conformal to the outer dimensions of the first reactor layer of the microfluidic reactor device, the latter can be properly mounted in a stable, accurate and more over in a repetitive manner on the first stage component. This stability and accurate mounting ensure allow easy, fast, automated and reproducible vitrification of samples for cryo-EM applications.

[0034] Preferably, the first stage component is made from a thermal conductive material, for example selected from the group consisting of aluminium, bronze or copper.

[0035] In addition, in a further example the mounting stage according to the disclosure may comprise a second stage component for mounting on the first stage component. This form a proper enclosure for the microfluidic reactor device, further ensuring easy, fast, automated and reproducible vitrification of samples for cryo-EM applications.

[0036] Preferably, the second stage component is made from a material which is the same or similar to the material of the first stage component.

[0037] Alternatively, the first stage component and / or the second stage component are provided with through bores for accommodating contact pins for electrically connecting the control unit with the heating means and / or the at least one pair of electrodes. This ensures a proper electrical connection of the various heating and detection means with the control unit further improving its feasibility for performing fast, automated and reproducible vitrification of samples for cryo-EM applications.

[0038] The disclosure also pertains to a method of analysing a sample under cryogen temperature conditions in a cryo-electron microscope. It comprises the subsequent steps of: i) providing a mounting stage according to the disclosure; ii) mounting the first reactor layer of a microfluidic reactor device according to the disclosure, with its second surface layer side on the first stage component; iii) applying a sample to be analysed on the reactor surface of the first surface layer side of the first reactor layer; iv) supplying - with the heating means - heat towards the reactor surface such that a temperature of at least the sample is raised to a first temperature; v) bringing the first stage component of the mounting stage in thermal contact with a cryogen, such that a temperature of at least the first reactor layer is lowered to a second temperature lower than the first temperature, thereby creating a thermal equilibrium between the first stage component and the first reactor layer; vi) interrupting the supply of heat as in step iv) whilst vii) maintaining the thermal contact of the first stage component of the mounting stage with the cryogen; and as an optional step, viii) monitoring - with at least one pair of electrodes structured - over time one or more material characteristics of the sample accommodated on the reactor surface.

[0039] By setting the heating means to a desired temperature, e.g. room temperature or body temperature, whilst bringing the first stage component in thermal contact with a cryogen, the first reactor layer containing the sample to be analysed is slowly cooled down to a cryogenic temperature, while the heating means maintain the sample at the desired temperature. The method according to the disclosure allows an user to optically inspect the sample via microscopy until the desired vitrification condition, at which moment the heating means are turned off. Due to the low thermal mass of the heating means and the sample near instantaneous freezing of the sample will occur, which process can be effectively monitored using the pair of electrodes.

[0040] In particular the above method step viii) involves the further steps of viii-1) detecting over time a change of impedance between the at least one pair of electrodes and viii-2) determining the one or more material characteristics of the sample accommodated on the reactor surface based on the change of impedance thus detected.

[0041] The method according to the disclosure allows the easiest, fastest, most automated and reproducible vitrification of samples for cryo-EM applications, if the first temperature is a temperature of above 0 °C, preferably a temperature in the range between 15 °C and 50 °C, more preferably a temperature in the range between 20 °C and 25 °C or a temperature in the range between 35 °C and 45 °C, even more preferably a temperature about room temperature or about the body temperature of the animal subject, preferably a human subject; and / or wherein the temperature is a temperature of below 0 °C, preferably a temperature below -100 °C, more preferably a temperature below -140 °C.

[0042] The method further comprises, after step viii), the step of repeating steps iv)-v)-vi)-vii)-viii) two or more times, thereby subjecting the sample to be analysed to two or more heating-cooling-monitoring cycles. This ensures reproducible vitrification of the same sample.

[0043] In particular, the cryogen cooling step v) and / or vi) involves a cooling rate of at least 1x106K / s, preferably a cooling rate of at least 1x107K / s, even more preferably a cooling rate of about 1x108K / s or more.

[0044] Preferably, the cryogen is selected from the group consisting of liquid nitrogen, liquid ethane, helium and slush nitrogen.

[0045] BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The disclosure will now be discussed with reference to the drawings, which show in:

[0047] Figures 1, 2A-2B and 3 various examples of a microfluidic reactor device according to the disclosure;

[0048] Figures 4A-4C various examples of a pair of electrodes to be implemented in microfluidic reactor device according to the disclosure;

[0049] Figures 5A-5E various functional views of an example of a mounting stage according to the disclosure suitable for mounting a microfluidic reactor device according to the disclosure;

[0050] Figures 6A-6F various side views of the example of a mounting stage according to the disclosure suitable for mounting a microfluidic reactor device according to the disclosure as shown in Figures 5A-5F;

[0051] Figures 7A and 7B a further detailed example of the electrode circuitry of a microfluidic reactor device according to the disclosure;

[0052] Figures 8A an experimental application of a temperature vs time graph outlining the flash freezing method according to the disclosure;

[0053] Figure 8B a simulation of another temperature vs time graph outlining the flash freezing method according to the disclosure.

[0054] DETAILED DESCRIPTION OF THE DISCLOSURE

[0055] For a proper understanding of the disclosure, in the detailed description below corresponding elements or parts of the disclosure will be denoted with identical reference numerals in the drawings.

[0056] Since the introduction of cryo-EM the sample preparation and vitrification has remained unchanged. All the vitrification methods require the use of specifically designed surface platforms and a cryogen. After sample deposition (typically drop-casted) on the analysis surface, the latter is either plunged in the cryogen or the cryogen is sprayed / jetted on the analysis surface to achieve vitreous ice. Current vitrification machines that use such analysis surfaces are extremely costly and complex with a large footprint.

[0057] Figures 1 , 2A-2B and 3 show various examples of an improved microfluidic reactor device denoted with reference numerals 10i , IO2, IO3 and IO4, that can be used in an efficient and reproducible manner in a cryo-electron microscope application, denoted in the drawings with reference numeral 1000, and has a less complex design and that reduces the size and the price of the vitrification machine.

[0058] The first example is shown in Figure 1. The microfluidic reactor device 10i can be used in a cryo-electron microscope 1000 and is of the open chip design. Such design can be implemented as a sample carrier embodied as a MEMS-based device. It is provided with one reactor layer 11 , which is at least partly transparent to an electron beam 1001 of the cryo-electron microscope 1000. The reactor layer 11 has a first surface layer side 11-1 and a second surface layer side 11-2 opposite from the first surface layer side 11-1. A part of the first surface layer side 11-1 is denoted with reference numeral 13 and is structured - during use - to function as a reactor surface 13 for accommodating a sample to be analysed.

[0059] The sample is denoted with reference numeral 1 (see Figure 5C) and may be a liquid containing analytes to be analysed. All types of biomolecules (e.g. proteins, DNA, viruses, exosomes, etc) can be used for analysing purposes, but also those which enable cell-culturing to vitrify eukaryotic cells. These types can be used to enable cryoElectron Tomography after the appropriate cryo-vitrifi cation. Furthermore, aside of primarily being used for biological research, the technology of the present disclosure can also be used to vitrify other types of soft-matter for material science research (e.g. MOFS, electrolytes for battery research, etc). It will be clarified further in the description, yet the second surface layer side 11-2 is structured - during use - to be brought in thermal contact with a cryogen 200.

[0060] Reference numeral 14 denote heating means which are received in or on the first reactor layer 11. The heating means 14 may comprise a heating element, for example in a non-limiting example a heater coil. The heating element according to the disclosure may be manufactured from molybdenum (Mo). The heating means 14 are intended to heat at least the reactor surface 13 of the first surface layer side 11-1.

[0061] Moreover, at least one pair of electrodes 15 are implemented that are structured - during use - to monitor material characteristics of the sample 1 accommodated on the reactor surface 13. In this example, the pair of electrodes 15 comprises at least two separate electrodes denoted with 15a and 15b. It is noted that the configuration of having at least one pair of electrodes 15 is an alternative example. It is noted that the microfluidic reactor device 10i according to the present disclosure may also function without at least one pair of electrodes 15.

[0062] In particular advantageous example, the at least one pair of electrodes 15 (15a-15b) are mounted in or on the first reactor layer 11 , optionally at least partially encapsulated in the first reactor layer 11 , or more optionally completely encapsulated in the first reactor layer 11. This ensures an effective measurement contact between the electrode pair 15 and the sample 1 to be analysed as being present directly above the pair of electrodes on the reactor surface 13 of the first reactor layer 11 .

[0063] A first example of the pair of electrodes 15i is shown in Figure 4A. In that example, the electrodes 15ai-15bi are each configured as single segment electrodes positioned at a certain distance d from each other. This allows for a fast position dependent measurement technique. In a more sophisticated example, shown in Figure 4B, allowing accurate monitoring and measurement of the various material or cryogenic characteristics over a sufficiently large contact area, the at least one pair of electrodes 152 are each configured as multi-segment electrodes 15a2 and 15b2. The multi-segment electrodes 15a2 and 15b2 each have multiple first and second extension elements 15a-x and 15b-x, which extension elements intermesh with each other.

[0064] In a further example as depicted in Figure 4C, the at least one pair of multisegment electrodes 15s are composed of first and second electrodes 15a3and 15bs having various extension elements 15a-x / 15a-y and 15b-x / 15b-y which are concentrically positioned with respect to each other.

[0065] Due to the extension of the at least one pair of electrodes 152 and 15s across the reactor surface 13 of the first reactor layer 11 an improved detection efficacy is ensured and a more accurate monitoring and measurement of the various material (cryogenic) characteristics over a sufficiently large contact area is achieved.

[0066] Apart from monitoring and detecting certain cryogenic characteristics, the at least one pair of electrodes 15 can also be used to fine-tune the wettability (contact angle) of the reactor surface 13 of first reactor layer 11 via electrowetting. Moreover, with the application of an electric field via the at least one pair of electrodes 15, any proteins (or any other type of biomolecule) in the sample 1 can be manipulated during the sample deposition on the reactor surface 13. This manipulation allows for electrophoresis to separate the particles (e.g., by weight / size), to prevent particle agglomeration, or to rotate the proteins in the sample 1 and align them with the electric field being applied between the electrodes 15. Figure 2A depicts a second example of a microfluidic reactor device IO2 according to the disclosure. In this example, the microfluidic reactor device IO2 comprises a second reactor layer 12. The second reactor layer 12 is like the first reactor layer 11 at least partly transparent to the electron beam 1001 of the cryo-electron microscope 1000. The second reactor layer 12 has a first surface layer side 12-1 that is facing the reactor surface 13 on the first reactor layer 11. The second surface layer side 12-2 of the second reactor layer 12 is facing outwardly towards the exterior, e.g. towards the cryo-electron microscope 1000.

[0067] The second reactor layer 12 extends next to the first reactor layer 11 at a mutual distance from each other. The gap between the first reactor layer 11 and the second reactor layer 12 forms together with proper reactor surface surrounding ridges or spacers 16a- 16b a closed reactor chamber 18. The reactor surface surrounding ridges or spacers 16a-16b are placed between the first reactor layer 11 and the second reactor layer 12 and function as a sealing spacer maintaining a stable and fixed gap between both layers 11-12. The inlet opening 17a and the outlet opening 17b are in fluid communication with each other via the reactor chamber 18, through which the sample can be fed and accumulated on the reactor surface 13 for analysing purposes.

[0068] Figure 2B shows an example nearly similar to the example of Figure 2A. However, in Figure 2B, the first reactor layer 11 of the microfluidic reactor device IO3 has a recessed part 11z in its second surface layer side 12-2. In the recessed part 11z heating connector contacts 140 and electrode connector contacts 150 are provided. The various heating connector contacts 140 (140-1 and 140-2) and the various electrode connector contacts 150 (150-1 and 150-2) are electrically connected through suitable micro-wiring 140z and 150z (see Figures 7A and 7B) with the heating means 14 and the pair of electrodes 15 (15a1-15a2, 15b1-15b2) respectively.

[0069] As will be explained later in the detailed description, the heating connector contacts 140 and the electrode connector contacts 150 can be electrically contacted suitable contact pins 174 and 175.

[0070] Additionally, as shown in Figure 2B, the first surface layer side 11-2 of the first reactor layer 11 is provided with a recess or cut-out 11q. The recess or cut-out 11q causes a local thinning of the first reactor layer 11 , which thinned part of the first reactor layer 11 serves as a transparent window for the electron beam 1001 thereby assisting in the optical visualization and inspection of the vitrification of a sample 1 present in and on the reactor surface 13 for cryo-EM applications.

[0071] The presence of a closed nano-channel or reactor chamber 18 formed between the reactor layers 11 and 12 eliminates any air-water interface problems during vitrification analysis. As shown, in Figures 1 , 2A-2B and 3 the inlet opening 17a and the outlet opening 17b are provided in and through the first reactor layer 11.

[0072] This compact design of the microfluidic reactor devices IO1-IO2-IO3-IO4 as shown in Figures 1 , 2A-2B and 3 will allow easy, fast, automated and reproducible vitrification of samples for cryo-EM applications. In addition, it will enable users to explore new techniques such as time-resolved cryo-EM. Furthermore, the integrated nano-sensors 15 (15a-15b) on the MEMS enables the user to immediately monitor various material characteristics, such as cryogenic characteristics e.g. the ice quality of the vitrified sample (e.g. thickness of the ice, crystallinity, etc). This will prevent users from proceeding with a faulty batch and wasting valuable transmission electron microscopy (TEM) time.

[0073] Apart from monitoring and detecting certain cryogenic characteristics, the at least one pair of electrodes can also monitor other material characteristics of the sample, such as its wettability (contact angle) via electrowetting. Moreover with the application of an electric field via the at least one pair of electrodes any proteins in the sample can be manipulated during the sample deposition on the reactor surface in order to avoid agglomeration or to rotate of the proteins in the sample and align them with the electric field being applied between the electrodes.

[0074] Preferably, the parts of the first and / or second reactor layers 11 and 12 which are transparent to the electron beam 1001 (hence those parts of the two layers which are within the view or propagation path of the electron beam 1001) are at a mutual distance of on average less than 100 nanometres, more in particular less than 20 nanometres and preferably less than 10 nanometres.

[0075] In particular, the mutual distance between the first reactor layer 11 and the second reactor layer 12 is a few micrometres, wherein the reactor chamber 18 enclosed between the parts of the reactor layers has an exposed surface, measured approximately parallel to the parts of the reactor layers 11 and 12, which is less than 20 mm2, in particular less than 10 mm2, more in particular less than 5 mm2and preferably of the order of magnitude of 1 mm2.

[0076] In a preferred example, the first and second reactor layers 11 and 12 are gas tight and liquid tight, thus preventing any leakage and / or contamination from outside the microfluidic reactor device IO2-IO3-IO4 (shown in Figures 2A-2B or 3) into the reactor chamber 18.

[0077] In order to improve the vitrification analysis of a sample 1 present in and on the reactor surface 13 and within the reactor chamber 18, windows 11w and 12w respectively are provided in the first and second reactor layers 11 and 12, which windows 11w and 12w are transparent to the electron beam 1001. These windows 11w and 12w are denoted in Figures 1 , 2A-2B and 3 schematically with a thick dashed line placed on and in the outer (second) surface layer sides 11-2 and 12-2 of both reactor layers. Preferably at least one window 11w in the first reactor layer 11 is located opposite to a window 12w in the opposite second reactor layer 12. In particular the windows 11w-12w present in the respective reactor layer 11-12 is more transparent to the electron beam than the remaining part of the respective reactor layer. To this end, for instance, the reactor layer can have been thinned locally, thereby forming a recess whose bottom part is formed by the window, and which has an increased transparency to the electron beam.

[0078] Here, each window 11w-12w has a surface which is particularly small compared with the total reactor surface 13 of the reactor chamber 18, for instance a few square micrometres. Sample to be analysed on a micrometre or even nanometre scale can be provided on such a window part of the reactor surface 13 so that a still better image can be obtained.

[0079] In a further advantageous example, the microfluidic reactor device is formed a monolithic component IO4 as shown in Figure 3. This increases the control on the maximum thickness for the resulting vitreous ice layer.

[0080] Optionally, yet not necessarily, in Figures 1-2A-2B and 3, the first reactor layer 11 can be mounted with its second surface layer side 11-2 on a first, lower microfluidic substrate 50 that supports the first reactor layer 11 and ascertains a certain rigidity and protection to the fragile component of the first reactor layer 11 . The first reactor layer 11 may be provided with an inlet opening 17a and an outlet opening 17b for supplying using suitable known means a certain amount of sample fluid 1 on the reactor surface 13. Optionally, the first, lower microfluidic substrate 50 may be provided with a substrate inlet opening 50a and a substrate outlet opening 50b, which are in fluid communication with the inlet opening 17a and the outlet opening 17b, respectively.

[0081] Optionally, the first, lower microfluidic substrate 50 may be provided with a recess window 50z, exposing the second surface layer side 11-2 and the window 11w of the first reactor layer 11 and indirectly also the reactor surface 13 present on the opposite first surface layer side 11-1. The recess allows for an improved optical visualization and inspection of the vitrification of a sample 1 present in and on the reactor surface 13 for cryo- EM applications.

[0082] In addition, as shown in the examples of Figures 2A-2B, a second, top microfluidic substrate 51 can be mounted on outer, second surface layer side 12-2 of the second reactor layer 12, thereby ascertaining additional rigidity and protection to the fragile components of the microfluidic devices IO2 and IO3, in particular that of the second reactor layer 12. The second, top microfluidic substrate 51 is either at least partly transparent to the electron beam 1001, however in a preferred example as shown in 2A and 2B a recess window 51 z is provided in the second, top microfluidic substrate 51 , exposing the reactor chamber 18 between the two reactor layers 11 and 12 to the electron beam 1001. With this configuration, the optical visualization and inspection of the vitrification of a sample 1 present in and on the reactor surface 13 for the purpose of cryo-EM applications is facilitated. This configuration could be particularly useful for users wanting to work with eukaryotic cells or big particles. Sealing 20 can be provided between both first and second microfluidic substrates 50-51 to ascertain a proper sealing between the first and second reactor layers 11-12 and thus the reactor chamber 18.

[0083] In particular, the at least one pair of sensing electrodes 15a- 15b are made of Au, Pt or Glassy Carbon. Optionally, the electrodes 15a-15b may be provided with an outer adhesion layer made of Ta or Ti, which improves adhesion to the first reactor layer 11 , in particular in the example that the first reactor layer 11 is made from of SiNx.

[0084] In the example of fully encapsulated electrodes, the pair of electrodes 15a- 15b may be made of Molybdenum (Mo) similar as the material of which the heating means (heating element or heating coil 14) is made of. That will simplify the fabrication.

[0085] A typical thickness of the electrodes 15a- 15b ranges between 100 nm and 250 nm, with a length of approx. 20 pm long and a width of approx. 10 pm, with a spacing of 1 pm distance between the pair of electrodes 15a- 15b.

[0086] Figures 5A-5E combined with corresponding side views of Figures 6A-6F shown another aspect of the present disclosure. Reference numeral 100 in Figures 5A-5E, 6A-6F denotes a mounting stage for use in a cryo-electron microscope. The stage 100 is designed to mount at least one microfluidic reactor device IO1-IO2-IO3-IO4 as outlined above in connection with Figures 1 , 2A-2B, 3 and 4A-4C. The mounting stage 100 is formed of at least a first stage component 101 for accommodating the first reactor layer 11 of the microfluidic reactor device IO1-IO2-IO3-IO4.

[0087] Figures 5A-5E show the mounting stage 100 having various first stage components 101, each structured to accommodate one of the examples of the microfluidic reactor device IO1-IO2-IO3-IO4 of Figures 1 , 2A, 2B or 3, whereas Figures 6A-6F depict cross sections of one of such first stage components 101. The one or more first stage components 101 are mounted to a base stage 110. Base stage 110 and the one or more first stage components 101 are preferably formed as a monolithic component or block made from the same, thermal conductive material, for example selected from the group consisting of aluminium, bronze or copper.

[0088] Each first stage component 101 is designed to be brought in thermal contact with a cryogen, denoted with reference numeral 200 in Figures 6E and 6F, either directly or indirectly via the base stage 110.

[0089] The first stage component 110 is provided with a recess 101z (see Figure 5A and 6A) for accommodating the first reactor layer 11 of the microfluidic reactor device IO1-IO2-IO3-IO4. For aligning purposes the first stage component 110 can be provided with an alignment notch 101q that erects from the recess 101z, thereby dividing the recess 101z in recess regions 101z-1 and 101z-2. The alignment notch 101 q accommodates the recess window 50z of the first, lower microfluidic substrate 50 supporting the first reactor layer 11. By providing the notch 101q in an asymmetrical fashion within the recess 101z, recess regions 101z-1 and 101z-2 are created having different dimensional sizes thus ascertaining the positioning of the microfluidic reactor device IO1-IO2-IO3-IO4 in only one correct orientation on the first stage component 110, thus avoiding a misalignment and an incorrect cryo-EM application.

[0090] By designing the recess 101z with an inner dimension conformal to the outer dimensions of the first reactor layer 11 or the outer dimensions of part of the first, lower microfluidic substrate 50 of the microfluidic reactor device IO1-IO2-IO3-IO4, the latter can be properly mounted in a stable, accurate and more over in a repetitive manner on the first stage component 101. This stability and accurate mounting ensure allow easy, fast, automated and reproducible vitrification of samples for cryo-EM applications.

[0091] The first stage component 101 is furthermore provided with multiple mounting bores 101a-1 , 101a-2 and 101 b-1 , 101b-2, preferably at the outer periphery of the first stage component 101, its functionality will be explained further in the detailed description.

[0092] In Figure 5B and 5C, any example of the microfluidic reactor device IO1-IO2- IO3-IO4 can be mounted within the recess 101z of the first mounting stage. In Figure 5B combined with Figure 6B, any example of the closed nano-channel design of microfluidic reactor device IO2-IO3-IO4 can be mounted. In Figure 5C, the open chip design of microfluidic reactor device 10i is mounted and provided with a certain amount sample 1 placed on the reactor surface 13 in the form of a droplet (through drop-casting).

[0093] In Figure 5D combined with Figure 6C, reference numeral 102 denotes a second stage component, which is suited to be mounted on the first stage component 101. This sandwich construction forms a proper enclosure for the microfluidic reactor device, further ensuring easy, fast, automated and reproducible vitrification of samples for cryo-EM applications.

[0094] In an example but not necessarily, the second stage component 102 is made from a thermal conductive material which is the same or similar to the material of the first stage component 101. In this configuration, in the situation that the first stage component 101 , yet the base platform 110 is brought in thermal contact with a cryogen 200, both the first stage component 101 as well as the second stage component 102 will be brought to the same cryogenic temperature, and hence the whole microfluidic device enclosed between both stage components 101-102 will likewise adopt the same cryogenic temperature. The later situation may not be desirable for certain cryo-EM applications.

[0095] The second stage component 102 is similarly provided with multiple mounting bores 102a-1 , 102a-2 and 102b-1 , 102b-2 provided at the outer periphery of the stage component 102, which mounting bores align with the mounting bores 101a-1 , 101a- 2 and 101 b-1 , 101 b-2 provided in the first stage component 101 (Figure 5D). Although not shown in Figure 6C, suitable mounting screws can be placed through the co-aligned mounting bores 101 b-1 / 102b- 1 and 101b- 2 / 102b-2, with the screw thread of the mounting screws interacting with corresponding internal screw threads provided within the co-aligned mounting bores 101 b-1 / 102b-1 and 101b- 2 / 102b-2. Herewith a proper fixation of the microfluidic reactor device IO1-IO2-IO3-IO4 within the mounting stage 100 is achieved.

[0096] Alternatively, an additional bore 102c may be provided in the second stage component 102, which exposes the heating connector contacts 140 and the electrode connector contacts 150 in the recessed part 11 z of the microfluidic reactor device 103 being accommodated between both first and second stage components 101-102 (see Figure 6C).

[0097] Similarly, the second stage component 102 whilst covering the microfluidic reactor device IO1-IO2-IO3-IO4 being accommodated in between, may be made of a material at least partly transparent to the electron beam 1001 of the cryo-electron microscope 1000. Alternatively, as shown in Figure 6C, the second stage component 102 may be provided with an opening or viewing window 102z, which exposes the viewing window 51z of the second, top microfluidic substrate 51 and the reactor chamber 18 to the electron beam 1001 , thus facilitating the optical visualization and inspection of the vitrification of a sample 1 present in and on the reactor surface 13 for the purpose of cryo- EM applications.

[0098] In Figure 5E and Figure 6D, reference numeral 103 denotes a third stage component or connector component for mounting on the second stage component 102. The connector component 103 is provided with multiple mounting bores 103a-1 and 103a- 2. Thes mounting bores each align with the corresponding mounting bores 102a-1 / 102a-2 of the second stage component 102 and the corresponding mounting bores 101a-1 / 101a- 2 provided in the first stage component 101 (see Figure 6D).

[0099] Although not shown in Figure 6D, suitable mounting screws can be placed through the co-aligned mounting bores 101a-1 / 102a-1 / 103a-1 and 101a-2 / 102a-2 / 103a-2, with the screw thread of the mounting screws interacting with corresponding internal screw threads provided within the co-aligned mounting bores 101a-1 / 102a-1 / 103a-1 and 101a- 2 / 102a-2 / 103a-2. Herewith a proper fixation of the third connector component 103 on the first stage component 101 is achieved.

[0100] Similarly, the third connector component 103 whilst mounted to the second stage component 102 may be made of a material at least partly transparent to the electron beam 1001 of the cryo-electron microscope 1000. The material is for example a non- conductive material selected from the group consisting of polymers, such as PMMA or PEEK. Alternatively, as shown in Figure 6D, it may be provided with an opening or viewing window 103z, which aligns with the viewing windows 102z of the second stage component 102 and likewise exposes the viewing window 51 z of the second, top microfluidic substrate 51 and the reactor chamber 18 to the electron beam 1001. Similarly, this also facilitates the optical visualization and inspection of the vitrification of a sample 1 present in and on the reactor surface 13 for the purpose of cryo-EM applications.

[0101] Reference numeral 103c denotes an additional contact bore, which aligns with the additional bore 102c provided in the second stage component 102. Both aligned additional bores 103c and 102c expose the heating connector contacts 140 and the electrode connector contacts 150 in the recessed part 11 z of the microfluidic reactor device IO3. The heating connector contacts 140 (140-1 and 140-2) and the electrode connector contacts 150 (150-1 and 150-2) can be electrically interconnected with conductive contact pins 174 and 175 respectively, see Figures 5E and 6E. The contact pins 174 and 175 reach into the co-aligned bores 103c and 102c and create an electrical contact with the respective heating connector contacts 140 and the electrode connector contacts 150. The contact pins 174 and 175 which are exposed from the connector component 103 are suited to electrically connected to a control unit 180 for controlling the heating means 14 and for reading out the at least one pair of electrodes 15 for acquiring the relevant information pertaining the materials characteristics of the sample 1 accommodated on the reactor surface 13. This configuration is highly feasible in performing fast, automated and reproducible vitrification of samples for cryo-EM applications.

[0102] In Figure 6F at least the first mounting stage 101 is provided with a supply port I inlet port 101x and a discharge port I outlet port 101 y, which are in fluid connection with the substrate inlet opening 50a I the inlet opening 17a and the substrate outlet opening 50b I the outlet opening 17b, respectively, through which the sample can be fed and accumulated on the reactor surface 13 for analysing purposes. In this example, proper sealings 111 are provided between the microfluidic reactor device 10nand the recess 101z of the first stage component 101.

[0103] The disclosure also pertains to a method of analysing a sample under cryogen temperature conditions in a cryo-electron microscope 1000. The method is closely linked to the mounting stage 100 discussed above and comprises the subsequent steps of: i) providing the mounting stage 100 according to the disclosure; ii) mounting the first reactor layer 11 of a microfluidic reactor device 101-IO2- IO3-IO4 with its second surface layer side 11-2 on the first stage component 110; iii) applying a sample 1 to be analysed on the reactor surface 13 of the first surface layer side 11-2 of the first reactor layer 11 either through e.g. drop-casting when implementing the open chip design of microfluidic reactor device 10i or through the fluid connection formed by the supply port / inlet port 101x, the substrate inlet opening 50a, the inlet opening 17a, the reactor chamber 18 and the outlet opening 17b, the substrate outlet opening 50b and the discharge port / outlet port 101y when implementing the closed chamber design of microfluidic reactor devices IO2-IO3-IO4; iv) supplying - with the heating means 14 - heat towards the reactor surface 13 such that a temperature of at least the sample 1 is raised to a first temperature; v) bringing the first stage component 101 of the mounting stage 100 in thermal contact with a cryogen 200, such that a temperature of at least the first reactor layer 11 of the microfluidic reactor device is lowered to a second temperature lower than the first temperature, thereby creating a thermal equilibrium between the first stage component 101 and the first reactor layer 11 ; vi) reducing or interrupting the supply of heat as in step iv) towards the sample 1 whilst vii) maintaining the thermal contact of the first stage component 101 of the mounting stage 100 with the cryogen 200; and as an optional step viii) monitoring - with the at least one pair of electrodes 15 - over time one or more material characteristics of the sample 1 accommodated on the reactor surface 13.

[0104] By setting the heating means 14 to a desired temperature, e.g. room temperature or body temperature, whilst bringing the first stage component 101 in thermal contact with the cryogen 200, the first reactor layer 11 containing the sample 1 to be analysed is slowly cooled down to a cryogenic temperature, while the heating means 14 maintain the sample 1 at the desired (room or body) temperature through continuous supply of heat towards the sample. The method according to the disclosure allows an user to optically inspect the sample 1 via microscopy using the electron microscope 1000 through the windows 103z-102z-51z and the electron beam transparent first reactor layer 11 (and optionally the second reactor layer 12) until the desired vitrification condition is achieved, at which moment the heating means are reduced or nearly turned off (but not completely).

[0105] Due to the close proximity of the first mounting stage 101 , which is held at a cryogenic temperature, the reducing of or practically turning down (interrupting) of the heating means, causes near instantaneous freezing of the sample 1 due to the low thermal mass of the heating means 14 and the sample 1. This instantaneous freezing process of the sample 1 can be effectively monitored using the pair of electrodes 15 as step viii) of the method according to the disclosure involves the further steps of viii-1) detecting over time a change of impedance (or resistance or capacitance) between the at least one pair of electrodes 15a- 15b.

[0106] In a further step viii-2) the one or more material characteristics of the sample 1 accommodated on the reactor surface 13 can be effectively determined based on the change of impedance '(or resistance or capacitance) thus detected by the electrodes 15a- 15b and the control unit 180.

[0107] The method according to the disclosure allows the easiest, fastest, most automated and reproducible vitrification of samples for cryo-EM applications, if the first temperature is a temperature of above 0 °C, preferably a temperature in the range between 15 °C and 50 °C, more preferably a temperature in the range between 20 °C and 25 °C or a temperature in the range between 35 °C and 45 °C, even more preferably a temperature about room temperature or about the body temperature of the animal subject, preferably a human subject; and / or wherein the temperature is a temperature of below 0 °C, preferably a temperature below -100 °C, more preferably a temperature below -140 °C.

[0108] The method further comprises, after step viii), the step of repeating steps iv)- v)-vi)-vii)-viii) two or more times, thereby subjecting the sample 1 to be analysed to two or more heating-cooling-monitoring cycles. Herewith the control unit 180 reactivates the heating means 14 causing the frozen sample 1 to heat and melt till the desired first (room or body) temperature. Subsequent reduction or practically turning down of the heating means 14 causes an anew near instantaneous freezing of the sample 1 due to the low thermal mass of the heating means 14 and the sample 1 , and the vitrification condition can optically be inspected again via microscopy and the one or more material characteristics of the sample 1 can be determined again using the change of impedance (or resistance or capacitance) detected by the electrodes 15a-15b and the control unit 180. This ensures reproducible vitrification of the same sample.

[0109] In particular, the cryogen cooling step v) and / or vi) involves a cooling rate of at least 1x106K / s, preferably a cooling rate of at least 1x107K / s, even more preferably a cooling rate of about 1x108K / s or more.

[0110] Preferably, the cryogen is selected from the group consisting of liquid nitrogen, liquid ethane, helium and slush nitrogen.

[0111] LIST OF REFERENCE NUMERALS USED

[0112] I sample to be analysed

[0113] 10, 10nmicrofluidic reactor device (first, second, third and fourth example)

[0114] 10’ monolithic microfluidic reactor device (fourth example)

[0115] I I first reactor layer

[0116] 11-1 first surface layer side of first reactor layer

[0117] 11-2 second surface layer side of first reactor layer

[0118] 11 q e-transparent window in first reactor layer

[0119] 11z connector recess in first reactor layer

[0120] 12 second reactor layer

[0121] 12-1 first surface layer side of second reactor layer

[0122] 12-2 second surface layer side of second reactor layer

[0123] 13 reactor surface

[0124] 14 heating means

[0125] 140 heating connector contacts

[0126] 140z micro-wiring of heating connector contacts

[0127] 15, 15npair of electrodes (first, second, third example)

[0128] 150 electrode connector contacts

[0129] 150z micro-wiring of electrode connector contacts

[0130] 15a, 15anfirst electrode (first, second, third example)

[0131] 15a-x first segment of first electrode (second, third example)

[0132] 15a-y second segment of first electrode (third example)

[0133] 15b, 15bnfirst electrode (first, second, third example)

[0134] 15b-x first segment of second electrode (second, third example)

[0135] 15b-y second segment of second electrode (third example)

[0136] 16a- 16b reactor surface surrounding ridge I spacer

[0137] 17-1 inlet opening

[0138] 17-2 outlet opening

[0139] 18 reactor chamber

[0140] 20 first sealing

[0141] 50 first, lower microfluidic substrate

[0142] 50a substrate inlet opening

[0143] 50b substrate outlet opening

[0144] 50z recess window 51 second, top microfluidic substrate

[0145] 51 z recess window

[0146] 100 mounting stage

[0147] 101 first stage component

[0148] 101a-b mounting bore in first stage component 101q alignment notch

[0149] 101x supply port / inlet port

[0150] 101 y discharge port I outlet port

[0151] 101z first mounting recess

[0152] 101z-1 / 2 recess region

[0153] 102 second stage component

[0154] 102a-b-c mounting bores in second stage component 102z viewing window of second stage component

[0155] 103 third stage component I connector

[0156] 103a-c mounting bores in third stage component 103z viewing window of third stage component

[0157] 110 base stage

[0158] 111 second sealing

[0159] 174 contact pins for heating means

[0160] 175 contact pins for electrode pair

[0161] 180 control unit

[0162] 200 cryogen

[0163] 1000 cryo-electron microscope

[0164] 1001 electron beam of cryo-electron microscope

Claims

CLAIMS1. A microfluidic reactor device for use in a cryo-electron microscope, comprising at least a first reactor layer being at least partly transparent to an electron beam of the cryo-electron microscope, the first reactor layer having a first surface layer side and a second surface layer side opposite from the first surface layer side, wherein at least part of the first surface layer side is structured - during use - to function as a reactor surface for accommodating a sample to be analysed and wherein the second surface layer side is structured - during use - to be brought in thermal contact with a cryogen, heating means structured for heating at least the reactor surface of the first surface layer side as well as a second reactor layer being at least partly transparent to the electron beam of the cryo-electron microscope, the second reactor layer facing and extending next to the first reactor layer at a mutual distance from each other and between which a reactor chamber is formed.

2. The microfluidic reactor device according to claim 1 , further comprising at least one pair of electrodes structured - during use - to monitor materials characteristics of the sample accommodated on the reactor surface.

3. The microfluidic reactor device according to claim 2, wherein the at least one pair of electrodes are mounted in or on the first reactor layer.

4. The microfluidic reactor device according to claim 2 or 3, wherein the at least one pair of electrodes are at least partially encapsulated in the first reactor layer.

5. The microfluidic reactor device according to claim 4, wherein the at least one pair of electrodes are completely encapsulated in the first reactor layer.

6. The microfluidic reactor device according to any one of the claims 2 to 5, wherein the at least one pair of electrodes extend across the reactor surface of the first reactor layer.

7. The microfluidic reactor device according to any one of the claims 2 to 6, wherein the at least one pair of electrodes are each configured as single segment electrodes positioned at a certain distance from each other.

8. The microfluidic reactor device according to any one of the claims 2 to 6, wherein the at least one pair of electrodes are each configured as multi-segment electrodes, which multi-segment electrodes intermesh with each other.

9. The microfluidic reactor device according to claim 8, wherein the at least onepair of multi-segment electrodes are concentrically positioned with respect to each other.

10. The microfluidic reactor device according to any one of the claims 2 to 9, wherein the at least one pair of electrodes are made from Au or Pt or Mo.

11. The microfluidic reactor device according to any one of the claims 2 to 10, wherein the at least one pair of electrodes sized electrodes has a thickness ranging between 100 nm and 250 nm, with a length of approx. 20 pm and a width of approx. 10 pm, with a spacing of 1 pm distance between the pair of electrodes.

12. The microfluidic reactor device according to any one of the claims 1 to 11 , further comprising an inlet and an outlet for feeding the sample through the reactor chamber and across the reactor surface.

13. The microfluidic reactor device according to claim 1 , wherein at least the parts of the first and / or second reactor layers which are transparent to the electron beam are at a mutual distance of on average less than 100 micrometres, more in particular less than 20 micrometres and preferably less than 10 micrometres.

14. The microfluidic reactor device according to claim 13, wherein the mutual distance is a few micrometres, wherein the reactor chamber enclosed between the parts of the reactor layers has an exposed surface, measured approximately parallel to the parts of the reactor layers, which is less than 20 mm2, in particular less than 10 mm2, more in particular less than 5 mm2and preferably of the order of magnitude of 1 mm2.

15. The microfluidic reactor device according to any one of the preceding claims, wherein the first and second reactor layers are gas tight and liquid tight.

16. The microfluidic reactor device according to any one of the preceding claims, wherein, in the first and second reactor layers, windows are provided which are transparent to the electron beam, wherein at least one window in the first reactor layer is located opposite to a window in the opposite second reactor layer.

17. The microfluidic reactor device according to claim 16, wherein the windows in at least one reactor layer are formed as recesses in the respective reactor layer.

18. The microfluidic reactor device according to any one of the preceding claims, wherein the heating means comprise a heating element, in particular a heater coil, which heating element is received in or on the first reactor layer.

19. The microfluidic reactor device according to any one of the claims 1 to 18, wherein the microfluidic reactor device is formed a monolithic component.

20. A mounting stage structured to mount the microfluidic reactor device according to any one of the preceding claims, the mounting stage comprising at least a firststage component for accommodating the second reactor layer of the microfluidic reactor device, and structured to be brought in thermal contact with a cryogen, as well as a control unit for controlling the heating means.

21. The mounting stage according to claim 20, wherein the control unit is structured for detecting a resistance or capacitance between the at least one pair of electrodes and for determining one or more material characteristics of the sample accommodated on the reactor surface based on the resistance or capacitance thus detected.

22. The mounting stage according to any of the claims 20-21 , further comprising a second stage component for mounting on the first stage component.

23. The mounting stage according to any of the claims 20 - 22, wherein the first stage component and / or the second stage component is made from a thermal conductive material, for example selected from the group consisting of aluminium or bronze or copper.

24. The mounting stage according to any of the claims 20-23, further comprising a third stage component for mounting on the second stage component.

25. The mounting stage according to claim 24, wherein the third stage component is made from a material being at least partly transparent to the electron beam of the cryo-electron microscope, for example selected from the group consisting of polymers, such as PMMA.

26. The mounting stage according to any one of the claims 20 to 25, wherein the first stage component and / or the second stage component are provided with through bores for accommodating contact pins for electrically connecting the control unit with the heating means and / or the at least one pair of electrodes.

27. A method of analysing a sample under cryogen temperature conditions in a cryo-electron microscope, comprising the subsequent steps of: i) providing a mounting stage according to any one or more of the claims 20 to 26; ii) mounting the first reactor layer of a microfluidic reactor device according to any one of the claims 1 to 19 with its second surface layer side on the first stage component; iii) applying a sample to be analysed on the reactor surface of the first surface layer side of the first reactor layer; iv) supplying - with the heating means - heat towards the reactor surface such that a temperature of at least the sample is raised to a first temperature; v) bringing the first stage component of the mounting stage in thermal contact with a cryogen, such that a temperature of at least the first reactor layer is lowered to asecond temperature lower than the first temperature, thereby creating a thermal equilibrium between the first stage component and the first reactor layer; vi) interrupting the supply of heat as in step iv) whilst vii) maintaining the thermal contact of the first stage component of the mounting stage with the cryogen.

28. The method according to claim 27 further comprising step viii) of monitoring - with at least one pair of electrodes structured - over time one or more material characteristics of the sample accommodated on the reactor surface.

29. The method according to claim 28, wherein step viii) involves the step of viii-1) detecting over time a change of impedance between the at least one pair of electrodes and viii-2) determining the one or more material characteristics of the sample accommodated on the reactor surface based on the change of impedance thus detected.

30. The method according to any of the claims 27-29, wherein the first temperature is a temperature of above 0 °C, preferably a temperature in the range between 15 °C and 50 °C, more preferably a temperature in the range between 20 °C and 25 °C or a temperature in the range between 35 °C and 45 °C, even more preferably a temperature about room temperature or about body temperature; and / or wherein the temperature is a temperature of below 0 °C, preferably a temperature below -100 °C, more preferably a temperature below -140 °C.

31. The method according to any one of the claims 27 to 30, further comprising, after step vii) or step viii), the step of repeating steps iv)-v)-vi)-vii)-viii) two or more times, thereby subjecting the sample to be analysed to two or more heating-cooling-monitoring cycles.

32. The method according to any one of the claims 27 to 31 , wherein the cryogen cooling step v) and / or vi) involves a cooling rate of at least 1x106K / s, preferably a cooling rate of at least 1x107K / s, even more preferably a cooling rate of about 1x108K / s or more.

33. The method according to any one of the claims 27 to 32, wherein the cryogen is selected from the group consisting of liquid nitrogen, liquid ethane, helium and slush nitrogen.

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