Method for preparing cryogenic samples with improved cooling properties

Abstract

To provide an improved method for preparing a cryogenic sample by vitrification.SOLUTION: The invention relates to a method and an apparatus for preparing a cryogenic sample, whereby the sample is subjected to rapid cooling using cryogenic fluid. A pair of conduits for transporting the cryogenic fluid are provided, each of the conduits opens into a mouthpiece, where the mouthpieces are arranged to face each other via an intervening gap, and where in the gap, the sample that is provided on a substantially planar sample carrier can be received. The cryogenic fluid can be pumped through the conduits so as to concurrently flush from the mouthpieces and suddenly immerse the sample in the cryogenic fluid from two opposite sides. As defined herein, at least one of the mouthpieces comprises at least two nozzle openings for evenly cooling the substantially planar sample carrier during flushing.SELECTED DRAWING: Figure 5A

Description

[Technical Field]

[0001] overview The present invention relates to a method for preparing a cryogenic sample, the sample being subjected to rapid cooling using a cryogen, the method comprising the steps of providing the sample on a substantially planar sample carrier; providing two conduits, each for transporting a cryogenic fluid, each of the conduits opening into a mouthpiece, the mouthpieces being positioned opposite each other across an intervening gap; and placing the sample carrier in the gap and pumping the cryogenic fluid through the conduits so that it flushes simultaneously from the mouthpieces, thereby rapidly immersing the sample in the cryogenic fluid from two opposite sides.

[0002] The invention further relates to an apparatus for carrying out such a method. [Background technology]

[0003] The term "cryogen" should be taken to refer to a liquid at cryogenic temperatures, i.e., at cryogenic temperatures below -150°C. Examples of such cryogens include liquid ethane, liquid propane, liquid oxygen, and mixtures thereof.

[0004] Charged particle microscopy is a well-known and increasingly important technique for imaging microscopic objects, particularly in the form of electron microscopy. Historically, the basic type of electron microscope has evolved into many well-known instruments such as transmission electron microscopes (TEMs), scanning electron microscopes (SEMs), and scanning transmission electron microscopes (STEMs), as well as various auxiliary instruments such as so-called "dual-beam" tools (e.g., FIB-SEMs) that further use a "machining" focused ion beam (FIB) to enable auxiliary functions such as ion beam milling or ion beam induced deposition (IBID). More specifically, - In SEM, irradiation of a sample by a scanning electron beam promotes the emission of "auxiliary" radiation from the sample in the form of secondary electrons, backscattered electrons, X-rays and photoluminescence (infrared, visible and / or ultraviolet photons), e.g. one or more components of the emitted radiation bundle are detected and used for image storage purposes. In a -TEM, the electron beam used to irradiate the sample is chosen to be of sufficiently high energy to penetrate the sample (hence it is generally thinner than in an SEM sample), and the bundle of transmitted electrons emitted from the sample can be used to generate an image. When such a TEM is operated in scanning mode (hence becoming a STEM), the image is accumulated during the scanning motion of the irradiating electron beam.

[0005] As an alternative to the use of electrons as the illuminating beam, charged particle microscopy can also be performed using other species of charged particles. In this regard, the phrase "charged particle" should be interpreted broadly to include, for example, electrons, positive ions (e.g., Ga ions or He ions), negative ions, protons, and positrons.

[0006] It should be noted that in addition to imaging and performing (local) surface modifications (e.g., milling, etching, deposition, etc.), charged particle microscopes can also have other functions, such as performing spectroscopy, examining diffractograms, etc.

[0007] In all cases, the charged particle microscope (CPM) comprises at least the following components: -Radiation sources such as Schottky electron sources or ion guns. An illuminator serves to manipulate the "raw" radiation beam from a source and perform certain operations thereon, such as focusing, aberration reduction, cropping (at an aperture), filtering, etc. It generally comprises one or more (charged particle) lenses, and may also comprise other types of (particle) optical components. If desired, the illuminator may comprise a deflector system that can be activated to perform a scanning motion of its output beam over the sample to be investigated. - a sample holder in which the sample under investigation can be held and positioned (e.g. tilted and rotated). If desired, this holder can be moved to effect a scanning movement of the beam relative to the sample. Typically, such a sample holder is connected to a positioning system such as a mechanical stage. The holder may comprise means for maintaining the sample within a given (high or low) temperature range, and in the specific context of the present invention will typically comprise means for maintaining the sample at a cryogenic temperature. The detector (for detecting radiation emanating from the irradiated sample) can be simple or multiple / distributed in nature and can take many different forms depending on the radiation to be detected. Examples include photodiodes, CMOS detectors, CCD detectors, photovoltaic cells, X-ray detectors (such as silicon drift detectors and Si(Li) detectors), etc. In general, a CPM can be equipped with several different types of detectors, and the choice of detector can be guided by different circumstances.

[0008] In the case of a transmission microscope (such as a (S)TEM), the CPM comprises: - an imaging system that essentially captures charged particles through a sample (plane) and directs (focuses) them onto an analytical device such as a detection / imaging device, a spectroscopic device (e.g., an EELS module; EELS = electron energy loss spectroscopy), etc. Similar to the illumination device mentioned above, the imaging system can also perform other functions such as aberration mitigation, cropping, filtering, etc., and it generally comprises one or more charged particle lenses and / or other types of optical particle components.

[0009] In the following, the invention is frequently described, by way of example, in the specific context of electron microscopy, however such simplifications are intended for clarity / illustration purposes only and should not be construed as limiting.

[0010] Biological specimens (e.g., cells, cellular components, single-cell organisms) that must be stored and observed within a body of aqueous fluid (e.g., water, electrolytes, cellular fluid, plasma) can present significant challenges for testing with CPM due to the following: Aqueous liquids introduced into the (quasi-)vacuum environment of the CPM tend to start outgassing / boiling and thus degrade the sample. To prevent this, the sample (specimen + aqueous liquid) can first be frozen before being introduced into the vacuum. However, to prevent sample damage caused by the formation of (sharp) ice crystals, such freezing must generally be carried out very rapidly, with the aim of achieving sample vitrification (solidification into an amorphous, glass-like phase) without significant ice crystallization.

[0011] To facilitate such vitrification and to allow the specimen to be examined in a transmission CPM such as a TEM, the sample should be relatively thin (sheet-like), but still be able to be supported at its edges (so that the support means used do not significantly affect the transmission of the beam). For this purpose, grid-like holders (such as the so-called TEM Autogrid®) are typically made, in which perforated membranes (so-called "perforated carbon membranes") are spread, in whose perforations a small amount of sample can be held (by surface tension effects). See, for example, Figure 1.

[0012] The method described in the opening paragraph above is known from US2017 / 0169991A1 (also published as EP3179229A1) and US2019 / 0180974A1 (also published as EP3495798A1), which have the same inventors / assignees as the present application and are incorporated herein by reference. The techniques described in said documents represent a significant improvement over the prior art.

[0013] It is an object of the present invention to provide an improved method for preparing cryogenic samples by vitrification. In particular, it is an object of the present invention that such a method should produce more consistent results as well as more consistent results across samples compared to prior art techniques.

[0014] These and other objects are achieved by a method for preparing a cryogenic sample as defined in claim 1, in which the sample is subjected to rapid cooling using a cryogen. The method includes the steps of providing a sample on a substantially planar sample carrier, providing two conduits for transporting a cryogenic fluid, each of the conduits opening into a mouthpiece, the mouthpieces being positioned facing each other across an intervening gap, and placing the sample carrier with the sample in the gap. As defined herein, the method includes the step of pumping the cryogenic fluid through the conduits to simultaneously flush from the mouthpieces, thereby rapidly immersing the sample in the cryogenic fluid from two opposing sides.

[0015] According to this method, at least one of the mouthpieces includes at least two nozzle openings. The nozzle openings are positioned to more uniformly distribute the liquid cryogen to at least one side of the sample carrier. The nozzle openings allow for more uniform cooling of the substantially planar sample carrier during flushing. By providing at least two nozzle openings, jets emanating from the at least two nozzle openings can cool two separate portions of the sample carrier at once, thereby allowing for more uniform cooling of the entire surface of the sample carrier. This allows for simultaneous vitrification of two different portions of the sample carrier, resulting in improved sample quality compared to vitrifying the bulk of the sample carrier.

[0016] Two nozzle openings allow, for example, the peripheral portion of the sample carrier to be cooled at least simultaneously with the central portion of the sample carrier. This is advantageous because it allows sample vitrification to occur substantially simultaneously in the peripheral and central portions of the sample carrier. This is particularly true when the substantially planar sample carrier is pre-assembled with a mechanical profile to improve handling of the sample carrier. Such mechanical profiles are known per se, for example, in the form of so-called clip rings (also known as AutoGrids). Such mechanical profiles typically have relatively large dimensions (and therefore relatively large mass) compared to the substantially planar sample carrier. Due to their relatively large mass, the mechanical profile retains a lot of heat during cryogenic flushing. This can result in the sample carrier being relatively cooler compared to the mechanical profile, and in fact, heat transfer from the mechanical profile to the sample carrier can occur. To prevent this, the invention described herein provides at least two nozzle openings for uniform cooling of the substantially planar sample carrier during flushing. This results in improved vitrification of the sample, achieving the objectives defined herein.

[0017] EP3475681B1 discloses a method for preparing a sample for imaging under cryogenic conditions, in which a jet of liquid coolant is used to vitrify the sample first at the center of the sample carrier and then toward the edge of the sample carrier. However, this approach has been found to be less than ideal, as it does not allow for simultaneous cooling of at least the peripheral portion of the sample carrier with at least the central portion of the sample carrier. This can potentially result in a decrease in overall sample quality. In contrast, the invention described herein provides at least two nozzle openings for uniformly cooling a substantially planar sample carrier during flushing. This allows sample vitrification to occur more uniformly throughout the sample carrier, and the nozzle openings can be positioned to cool at least the peripheral portion of the sample carrier simultaneously with the central portion of the sample carrier. Additionally, at least two nozzles can be used to counteract the heat flux from the mechanical profile to the sample carrier, resulting in more uniform cooling and vitrification of the sample, even when a mechanical profile is used. Summary of the Invention

[0018] Advantageous embodiments are described below.

[0019] In one embodiment, the at least two nozzle openings comprise a central nozzle arranged to cool a central portion of the substantially planar sample carrier and at least a peripheral nozzle arranged to cool a peripheral portion of the substantially planar sample carrier. In this way, the periphery of the sample carrier can be cooled simultaneously with at least the central portion of the sample carrier. It is believed that the periphery of the sample carrier cools faster than the central portion of the sample carrier. This is advantageous when a mechanical profile is used. The use of a central nozzle and at least a peripheral nozzle allows the peripheral portion to cool slightly faster than the central portion, thereby counteracting the aforementioned heat transfer from the mechanical profile to the peripheral portion of the sample carrier. This contributes to more uniform cooling and vitrification of the sample on the sample carrier.

[0020] In one embodiment, at least two nozzle openings are arranged to provide substantially identical flushes of cryogenic fluid onto a substantially planar sample carrier, which flushes may be substantially identical with respect to one or more of temperature, coolant, flow rate, flow time, flow pressure, flow velocity, total flow rate, and flow size.

[0021] In another embodiment, at least two nozzle openings are arranged to provide a substantially non-uniform flush of cryogenic fluid onto a substantially planar sample carrier.

[0022] In one embodiment, the central nozzle is positioned to establish a lower flash of cryogenic fluid compared to at least one peripheral nozzle.

[0023] In one embodiment, the central nozzle and the peripheral nozzles are substantially identical in size. In this way, the area of ​​the sample carrier affected by each nozzle is substantially the same. In another embodiment, the central nozzle has smaller dimensions compared to at least one peripheral nozzle. This allows the peripheral nozzle to cool a little more, resulting in more uniform cooling and vitrification of the entire sample on the sample carrier.

[0024] At least one of the mouthpieces may include multiple peripheral nozzles. In particular, embodiments with two, three, four, or five peripheral nozzles provide more uniform cooling of the entire sample carrier. The peripheral nozzles may be evenly spaced around the mouthpiece, particularly around the central nozzle.

[0025] In one embodiment, the nozzle plate includes at least six peripheral nozzles. The six peripheral nozzles may be evenly spaced around a central nozzle. This arrangement has been found to provide superior results in terms of more uniform temperature distribution throughout the sample carrier during flushing with the cryogenic fluid. Each nozzle allows for more localized cooling of the sample carrier at a given time. The peripheral nozzles may be substantially the same size. The central nozzle may be the same size as the peripheral nozzles, or the central nozzle may be slightly smaller than the peripheral nozzles.

[0026] As defined herein, one of the mouthpieces includes at least two nozzle openings. The other of the two mouthpieces is also considered to include at least two nozzle openings. These at least two nozzle openings are hereinafter also referred to as "additional nozzle openings." The nozzle openings provide more uniform cooling on a first side of the sample carrier, and the additional nozzle openings provide more uniform cooling on the opposite side of the sample carrier. By providing a nozzle opening and an additional nozzle opening, more uniform cooling is possible on both sides of the sample carrier in a similar manner as described above with at least two nozzle openings. More uniform cooling can be provided, where the cooling can be equal on both sides of the sample carrier, or the cooling can be substantially different on both sides of the sample carrier.

[0027] In one embodiment, the further nozzle openings comprise a single central nozzle opening and six peripheral nozzle openings evenly distributed around the central nozzle opening. In this embodiment, two mouthpieces may be similarly embodied, i.e., each having a central nozzle opening and six peripheral nozzle openings, each nozzle opening having substantially the same dimensions.

[0028] The nozzle openings of a single mouthpiece can be directly connected to corresponding conduits, so that a single conduit can be used to transport cryogenic liquid to the available nozzle openings. However, each nozzle opening is considered to have its own conduit. Methods for providing cryogenic fluid to specific nozzle openings are known per se to those skilled in the art. In one embodiment, the mouthpiece can be formed as a nozzle plate covering a single conduit, and the nozzle openings are provided in the nozzle plate. This allows a single conduit to supply the desired cryogenic fluid to all nozzle openings of the nozzle plate.

[0029] According to this method, the flush of cryogenic fluid from the first mouthpiece is reduced after a given time interval such that the flush of cryogenic fluid from the first mouthpiece is different from that applied from the second mouthpiece, creating an intentional asymmetry / parallel difference in the cooling of the backside (on one side of the mouthpiece) and frontside (on the opposite side of the mouthpiece) of the sample.

[0030] In another embodiment, the flush of cryogenic fluid applied from the first mouthpiece is changed after a given time interval, such that the flush of cryogenic fluid from the first mouthpiece after the time interval is different from the flush of cryogenic fluid from the first mouthpiece before the time interval. In one embodiment, the flush from one of the nozzles is changed while the flush from another one of the nozzles remains the same. This can, for example, reduce or eliminate the flush from the central nozzle opening while keeping the flush from the peripheral nozzles constant. This can help protect the sample provided in the sample carrier.

[0031] In another embodiment, the flush of cryogenic fluid applied from the first mouthpiece is substantially identical to the flush of cryogenic fluid applied from the second mouthpiece.

[0032] For completeness, it should be recognized that the cryogenic fluid flushed from the mouthpieces may be liquid or (dry) gas / vapor and may be the same or different for each mouthpiece, with liquids generally tending to be preferred over gas-phase fluids due, among other things, to the liquid's greater heat capacity and relative ease of storage and pumping. It should also be noted that, if desired, one or more of the at least two nozzle openings may include a mesh (sieve) or other form of flow channel subdivision, for example, to achieve laminar flow and / or a particular flow pattern. As suggested above, this mesh need not be the same in both mouthpieces.

[0033] In an embodiment, the reduction includes terminating the flush from the first mouthpiece after a given time interval, so that one side is cooled for a longer period of time compared to the opposite side.

[0034] In a setup according to the invention, the cryogenic fluid can be pumped through the use conduits, for example, using one or more (electrical) pumps that can be switched on / off as needed and / or a valve system can be used to open and close the flow in the conduits at will. Such an arrangement allows, for example, the flow rate and / or flow time in one of the conduits (according to one embodiment of the invention) to be different from the flow rate and / or flow time in another conduit.

[0035] In an alternative embodiment, the conduits are placed within the plunger, whereby each conduit has an inlet opening on the underside of the plunger, the gap is provided as a slot on the top surface of the plunger, a reservoir of cryogenic fluid is provided below the plunger, and the sample is inserted into the slot using a tool that applies downward pressure on the plunger, thereby at least partially submerging the plunger and causing the cryogenic fluid in the reservoir to flow into the inlet opening and out via the mouthpiece, in a "hand pumped" setup.

[0036] Such a configuration effectively utilizes a fluid displacement mechanism similar to that used for pistons, with a piston plunger acting as the piston head (with an overflow conduit and a cryogenic bath contained in the piston tube). This is similar to the configuration described in the aforementioned US2017 / 0169991A1. The modifications described in the aforementioned US2019 / 0180974 can also be incorporated. These modifications allow for the generation of different flushes of cryogenic fluid from each of the mouthpieces. One way to achieve this is to use a shield to close a first of the conduits connected to the first mouthpiece after a given time interval. A convenient way to perform such delayed shielding is for the plunger to be submerged in the bath, with a first opening connected to the first conduit engaging a lid (cover, blanking member) disposed a selected distance d below the surface of the bath, and the lid co-moves with the plunger to keep the first opening closed. Such a scenario is shown, for example, in Figures 3A-3C. If the plunger is submerged at a vertical velocity v, d can be selected to engage the shield at a given time t=d / v (e.g., in the range of 10-100 ms, such as 40-60 ms) after the plunger starts flushing / depressing. The shield itself can be, for example, For example, the cryogen bath employed may be made inherently buoyant and / or artificially "buoyant" by, for example, biasing it upward with a spring mechanism, such that when the plunger disengages therefrom (on the up / return "stroke"), the shield returns upward (to its default / initial position). - Mounts on guides / rails to constrain motion to a vertical path. -An adjustable stopper is used to prevent the container from rising above a certain level (depth d).

[0037] As described in the previous paragraph, in this embodiment, it should be noted that: The "tool" in question may be, for example, tweezers or pliers, which can be used to grasp the end of the sample piece. Such a tool may include features such as a protrusion, bar, or local enlargement that engages with the top of the plunger (or some structure thereon) to translate, for example, the insertion motion of the tool (in the slot) into a co-movement (downward) of the plunger. The plunger may initially float (shallowly) in the cryogen bath, or alternatively, may hang above the bath. 3A-3C depict a scenario in which the sample plane is oriented vertically and the cryogenic fluid is flushed horizontally from the mouthpiece, but this need not be the case. Instead, for example, a configuration can be constructed in which the sample plane is oriented horizontally, with mouthpieces positioned above and below it, and the cryogenic fluid is flushed vertically from the mouthpiece. In both scenarios, it is desirable to have the (cumulative) lengths of both conduits substantially equal so that the cryogenic fluid is dispensed substantially synchronously from both mouthpieces when the plungers are suitably submerged. The plunger can be made from a variety of materials, as long as they are compatible (e.g., in terms of brittleness) for use at cryogenic temperatures. Examples include, for example, stainless steel, titanium, and (certain) ceramics.

[0038] In the case of grids / membranes as described above or substantially planar samples with oppositely positioned major surfaces, such as, for example, flakes / wafer pieces, the following considerations provide (non-binding) guidance when applying the present invention. Each mouthpiece can have an associated end / extreme / circumference, which is typically circular, but this does not necessarily have to be the case. These peripheries are ideally centered on a common axis. If a central nozzle opening is used, the central nozzle opening may be aligned with the common axis. -This common axis intersects the plane of the (planar) sample at an (approximately) perpendicular / orthogonal angle. The common axis ideally passes through the geometric centre / centre of mass / centre of gravity of the sample. -Nominally, the sample is positioned so that it is equidistant from the mouthpiece (or, in the case of samples that include membranes spread on a grid, so that the membranes are equidistant from the mouthpiece). If the sample / membrane is close to a particular mouthpiece, flushing of the front and back sides of the sample can be initiated simultaneously, for example: -Slightly delay the pumping of cryogenic fluid into the conduit connected to a particular mouthpiece and / or -Making the conduits connected to certain mouthpieces slightly longer than the conduits connected to other mouthpieces.

[0039] Regarding the cryogen bath in which the sample is submerged, there are various options regarding the cryogen used. For example, various practitioners have reported using a liquid ethane / propane mixture. In a specific embodiment of the present invention, the cryogenic fluid in the bath comprises liquid ethane (without substantial amounts of other components) at temperatures ranging from −160°C to −183°C. When vitrifying samples by plunge cooling, the “colder is better” rule tends to be followed. However, it has been observed that at temperatures below about −183°C, liquid ethane becomes very viscous and can interfere with the plunging process, such as by adhering to the sample holder. Therefore, temperatures above this level (e.g., −175°C) are generally preferred.

[0040] To achieve successful vitrification of the sample, exposure to the cryogenic fluid from the mouthpiece of the present invention must be relatively sudden, i.e., "flash." Using two nozzle openings on at least one of the mouthpieces helps ensure sudden exposure of the cryogenic fluid over an increased area of ​​the entire sample carrier. A more gradual / extended exposure to the cryogenic fluid from the mouthpiece risks freezing the sample (at least partially) into a crystalline form rather than amorphizing it, which is undesirable in the context of the present invention. The nozzle opening can be adjusted / optimized to establish the desired flow depending on various factors, such as the sample (grid / membrane) thickness, the temperature of the cryogenic fluid used, and the pressure / flow pattern generated by the nozzle opening of the mouthpiece. By way of non-limiting example, the uninterrupted flash from the at least two nozzle openings may have a nominal duration, e.g., in the range of 5 to 200 milliseconds. For samples containing a holey film on a grid, this may ensure sufficient removal of residual heat from the grid to prevent unwanted "reheating" of the film due to heat leakage from the grid or unwanted "reheating" of the sample carrier due to heat leakage from the mechanical support, in addition to producing vitrification of the aqueous film in the holey film. After vitrification occurs, the temperature of the aqueous film is preferably maintained below approximately -145°C to prevent unwanted crystalline ice formation. Those skilled in the art will be able to select a flash duration tailored to the configuration and parameters associated with a given embodiment of the present invention.

[0041] In one embodiment, flushing from both mouthpieces begins substantially simultaneously, but flushing from the first mouthpiece ends after a given time interval. In a further embodiment, a shield can be used to close off the first of the conduits connected to the first mouthpiece after a time interval.

[0042] In one embodiment, a plunger is submerged in the vessel, the first opening is connected to the first conduit, and engages a lid disposed at a selected distance below the surface of the vessel, the lid moving with the plunger to hold the first opening closed.

[0043] In one embodiment, the plunger is surrounded by at least one seal element to prevent leakage of the cryogenic fluid when downward pressure is applied to the plunger. The plunger may be surrounded by at least two seals. A second seal element may be positioned to open the discharge channel when the plunger is returned toward the start position.

[0044] In one embodiment, an overpressure can be provided to the reservoir below the plunger to ensure that the conduit fills with cryogenic fluid the moment the sample tool begins to push the plunger downwards.

[0045] According to one aspect, there is provided an apparatus for carrying out the method defined herein, the apparatus being defined according to claim 13. An apparatus for preparing a cryogenic fluid sample, whereby the sample is subjected to rapid cooling using a cryogenic fluid, the apparatus comprising: a pair of conduits for transporting a cryogenic fluid, each of the conduits opening into a mouthpiece, the mouthpieces being arranged opposite each other with an intervening gap in which a sample provided on a substantially planar sample carrier can be received; a pumping mechanism for pumping the cryogenic fluid through the conduits to simultaneously flush from the mouthpiece, rapidly immersing the sample in the cryogenic fluid from two opposing sides.

[0046] As defined herein, the device is characterized in that at least one of the mouthpieces comprises at least two nozzle openings for uniformly cooling the substantially planar sample carrier during flushing. The advantages of such a device have been elucidated in this way.

[0047] Further embodiments are the subject of the dependent claims. [Brief explanation of the drawings]

[0048] The invention will now be elucidated in detail on the basis of exemplary embodiments and the accompanying schematic drawings. [Figure 1A] FIG. 1 shows (a specific embodiment of) the general sample structure used in the vitrification procedure. [Figure 1B] FIG. 1 shows (a specific embodiment of) the general sample structure used in the vitrification procedure. [Figure 2] FIG. 1 illustrates sample damage that can occur by applying prior art vitrification procedures. [Figure 3A] 1 is a longitudinal section rendering of an embodiment of an apparatus according to the invention (for defining the method of the invention); FIG. [Figure 3B] 1 is a longitudinal section rendering of an embodiment of an apparatus according to the invention (for defining the method of the invention); FIG. [Figure 3C] 1 is a longitudinal section rendering of an embodiment of an apparatus according to the invention (for defining the method of the invention); FIG. [Figure 4] FIG. 1 depicts a longitudinal cross-sectional view of a charged particle microscope suitable for use in the present invention. [Figure 5A] FIG. 1 shows a schematic representation of an embodiment of a mouthpiece having at least two nozzle openings as defined herein. [Figure 5B] FIG. 1 shows a schematic representation of an embodiment of a mouthpiece having at least two nozzle openings as defined herein. [Figure 5C] FIG. 1 shows a schematic representation of an embodiment of a mouthpiece having at least two nozzle openings as defined herein. [Figure 5D] FIG. 1 shows a schematic representation of an embodiment of a mouthpiece having at least two nozzle openings as defined herein. [Figure 5E]FIG. 1 shows a schematic representation of an embodiment of a mouthpiece having at least two nozzle openings as defined herein. [Figure 5F] FIG. 1 shows a schematic representation of an embodiment of a mouthpiece having at least two nozzle openings as defined herein. [Figure 6] FIG. 1 illustrates a sample vitrified using the method and apparatus described herein. [Figure 7] 1 shows a further embodiment of the device according to the invention; DETAILED DESCRIPTION OF THE INVENTION

[0049] Corresponding parts may be indicated using corresponding reference symbols. It should be noted that the figures are generally not to scale.

[0050] Background example FIG. 1A renders a detailed (enlarged) overview of aspects of a particular embodiment of a sample S that can be used in conjunction with the present invention. This particular type of sample S includes what is often referred to as a "grid" G. It includes a circular ring of wire 21a (e.g., comprising Cu or Ni), the ring diameter typically being on the order of about 3 mm, and the wire diameter typically being on the order of about 20-100 μm. Attached within the ring 21a are straight wire segments 21b arranged (in this case) to form an orthogonal grid pattern, defining a matrix-like array of (approximately square) apertures (holes / windows) 23. The middle portion of FIG. 1A shows a cross-sectional view along diameter B-B' at the top of the figure. This shows that the grid G ​​has a substantially flat (plate-like) shape with first (S1) and second (S2) "faces" that are substantially parallel and opposite to each other. As shown here, a membrane 25 is spread across the first surface S1 (optionally secured to the wire 21b using, for example, an adhesive or fusion bonding). This membrane 25 comprises a carbonaceous material, such as nylon or graphene, and typically has a thickness (Y direction) ranging from about 0.3 nm to several hundred nanometers. The membrane 25 contains a distribution of perforations 27, which are clearly visible in the detailed view at the bottom of the figure. These perforations 27 typically have diameters (parallel to the XZ plane) ranging from about 1.2 to 3.5 μm (e.g., about 2 μm). Essentially, the grid G ​​serves as a scaffold for the membrane 25, which in turn acts as a support structure for the perforations 27 (sometimes referred to as a "porous carbon support"). Within the perforations 27, the final "sample" or "specimen" is provided and supported, in the form of a thin film 29 of aqueous liquid (containing one or more test specimens suspended therein) that is spread across each given perforation 27, held in place (among other things) by surface tension effects. A structure such as that depicted in FIG. 1A (Grid G+perforated membrane 25, 27) and described above is commercially available, for example, from Ted Pella, Inc. of Redding, Calif., USA.Furthermore, it is possible to purchase, for example, (various) pre-fabricated holey carbon films (corresponding to perforated membranes 25, 27) from companies such as, for example, Quantifoil Micro Tools GmbH, Jena, Germany. Notably, in the context of the present invention, the illustrated structure can be considered to have a "back side" Sb and a "front side" Sf.

[0051] The film 29 of aqueous liquid can be applied to the various perforations 27 of the membrane 25 using methods well described in the technical literature and known to those skilled in the art. In one such known method, a piece of absorbent paper (not shown) is pressed against the outer / lower surface of the membrane 25, then moistened with the aqueous liquid in question, and the membrane 25 is subsequently removed (e.g., peeled off), causing the (most) openings 27 to be provided with a (mini) film 29 of aqueous liquid, which is spread therein by surface tension effects. This type of method is described, for example, in the article "Electron Microscopy of Frozen Water and Aqueous Solutions" by J. Dubochet et al. in Journal of Microscopy, Vol. 128, pt. 3, December 1982, pp. 219-237, but will not receive further attention here. Reference is also made to an alternative method described in U.S. Pat. No. 9,772,265 (which has the same inventors / assignee as the present application and is incorporated herein by reference).

[0052] Turning now to FIG. 1B, an example of an EM grid sample S with a mechanical profile 31 for improved handling is shown. Here, the grid S depicted in FIG. 1A is substantially circular and surrounded by a first profile 31 with an L-shaped cross-sectional area. This first profile 31, also referred to as a clip ring 31 and known to those skilled in the art, is provided within a recess in the mechanical profile 31, abutting the recessed surface of the mechanical profile 31. A fixing element 33 in the form of a c-shaped clip 33 (also referred to as a c-clip 33) firmly holds the grid S in place within the recess in the mechanical profile 31. A thin film 29 of aqueous liquid containing the sample / specimen to be investigated is provided on the back or front side of the grid S (shown here in an exaggerated manner; the drawing is not to scale).

[0053] Referring now to Figure 2, this shows a particular sample of the type shown in Figures 1A and 1B after vitrification using the method described in the aforementioned US 2017 / 0169991 A1 (the same flash was applied to the back and front sides of the sample). The light gray squares / cells are undamaged after a successful vitrification procedure, while the white squares / cells are (more or less) damaged where membrane breakage / delamination has occurred. The dark / mottled squares / cells correspond to areas where vitrification has occurred, but where the ice is (more or less) too thick. It can be seen that approximately 25% of the squares / cells are suboptimal in the current situation. As noted above, closer examination of the sample (not evident in the current diagram) reveals that the damaged squares / cells are primarily oriented from the back to the front. In contrast, Figure 6 shows a particular sample of the type shown in Figures 1A and 1B after vitrification using the method described herein using the apparatus described herein. It can be seen that most of the squares / cells are white and therefore undamaged. Only a few cells show dark / mottled spots and significantly fewer squares are damaged.

[0054] Embodiment 1 Figures 3A-3C show aspects of an embodiment of the method and apparatus according to the present invention. Starting with Figure 3A, this shows a pair of conduits 31a, 31b for transporting a cryogenic fluid. Each of these conduits 31a, 31b has two ultimate orifices, which (respectively) - (lower) inlet orifices 37a, 37b through which the cryogenic fluid can enter the conduits 31a, 31b; and (upper) mouthpieces (exit orifices) 33a, 33b through which the cryogenic fluid can exit the conduits 31a, 31b. These mouthpieces 33a, 33b face each other with an intervening gap 35. The mouthpieces 33a, 33b are provided with nozzle plates 39a, each of which has at least two nozzle openings. These nozzle openings and their possible embodiments can be illustrated by Figures 5A to 5D.

[0055] The following should be noted: For convenience, the various components 31 a, 31 b, 33 a, 33 b, 35, 37 a, 37 b are shown here as being within a body P that may act as a matrix / structure to hold them in place, for example, body P being a metal, ceramic or epoxy plug / block, the various components being made by, for example, casting, molding, machining or 3D printing. However, this is not necessarily the case, and the various components may instead be (quasi-) free-standing structures. Although the orifices 37a, 37b; 33a, 33b are depicted here as flared, this need not be the case. The gap 35 is shown as being of uniform width, but may alternatively be tapered, for example, and should ideally be relatively narrow (in the Y direction) to improve synchronization and symmetry of the initial flash.

[0056] Also shown in Figure 3A is a tool T (such as tweezers, scissors, pliers, a clamp, a robotic arm, etc.) that can be used to grasp and manipulate sample S by grasping it along its edges, for example, as shown in Figure 1B by mechanical contour 31. This tool T can be used to position sample S within gap 35 and between mouthpieces 33a, 33b.

[0057] As already mentioned above, one way to supply cryogen to the inlet orifices 37a, 37b is to connect them to an (electric) cryogen pump (and associated cryogen reservoir) using suitable tubing / plumbing, and one can pump the cryogen through the conduits 31a, 31b and out of the mouthpieces 33a, 33b in order to flush / shower the gap 35 (the sample S located therein) with the cryogen. However, in this embodiment, instead, a (manual) piston action is used to move the cryogen through the conduits 31a, 31b. For this purpose, the body P is embodied as a plunger having a lower side Pu (where the inlet orifices 37a, 37b are located) and an upper side Pt (which is able to access the gap 35). This plunger P may, for example, be (partially) submerged / immersed in a container (tube, vessel) 5 of cryogen 7, and as the plunger's underside Pu moves below surface 9, the cryogen 7 is (progressively) forced through inlet orifices 37a, 37b, through conduits 31a, 31b, and out mouthpieces 33a, 33b (FIGS. 3A-3B-3C show part of this movement). Note in FIG. 3A that prior to the start of the tool T insertion / plunging procedure, conduits 31a, 31b are primed / pre-filled with cryogen 7, e.g., as a result of pre-siphoning and / or capillary action from previous plunging iterations. In this way, the cryogen is supplied in close proximity to the inserted location of sample S, ready to be instantly vented, reducing the risk of asynchronous flashing from both sides of sample S.

[0058] To generate the desired plunger motion, the illustrated configuration uses tool T to apply a downward force to plunger P, but this is not necessarily the case; plunger P can instead be pushed downward by other means. As shown in FIGS. 3B and 3C , tool T has a protrusion / lug T′ that engages a reciprocal region / portion P′ of the top surface Pt of plunger P, allowing a downward force on tool T to transfer downward momentum to plunger P—see the exemplary downward arrow T″ in FIGS. 3B and 3C . Furthermore, protrusion T′ can be utilized (if so desired) to ensure sample S is inserted to an optimal depth within gap 35 (ideally substantially symmetrically between mouthpieces 33 a, 33 b) and can also be used to provide correct lateral positioning of sample S within gap 35 (again, ideally with a (glassy film) equidistant from mouthpieces 33 a, 33 b).

[0059] In the non-limiting example of a configuration such as that shown here, the following exemplary (and approximate) values ​​may apply: The sample S comprises a planar grid with a diameter of 3 mm and a thickness of 0.4 mm. -Diameter of mouthpieces 33a and 33b: 3-4mm. - diameter of conduits 31a, 31b: 2.5 mm; Width of the separation / gap 35 between the mouthpieces 31a, 31b: 1 mm. -Flow rate from mouthpieces 31a and 31b: approximately 5 to 15 m / s.

[0060] Those skilled in the art will be able to adapt their own values ​​to the needs of a given situation.

[0061] 3A-3D, the illustrated device is configured such that the flush of cryogenic fluid delivered from (left) mouthpiece 33a is different from the flush delivered from (right) mouthpiece 33b, more specifically such that the duration of the flush from mouthpiece 33a is shorter than the duration of the flush from mouthpiece 33b. For this purpose, a blocking mechanism (41, 43, 45) is used which closes (left) conduit 31a (connected to (left) mouthpiece 33a) after a given time interval. More specifically, this blocking mechanism comprises: a lid (cover / blanker) 41 positioned at a selected nominal distance d below the surface 9 of the cryogen bath; The rails / guides 43 allow and guide movement of the lid 41 substantially parallel to the Z axis. To this end, the lid 41 may be provided with an opening 43' (or other suitable structure, such as, for example, a clasp or runner) that cooperates with the rails 43 to allow relative movement of the two. - An adjustable stop (for example a screwed anti-slip collar) that prevents the lid 41 from moving upwards beyond a certain point (depth d). As noted above, the lid 41 may naturally float in the cryogenic fluid 7 (e.g., because it is hollow) and / or may be biased upwards using, for example, a spring, piston, or magnetic arrangement. In this way, the lid 41 may move downwards with the plunger P when engaged, but return / slowly pull upwards when the plunger P disengages therefrom. With particular reference to the individual figures, In Figure 3B, such engagement has not yet occurred. The plunger P moves downwards through the cryogen 7, forcing (more) cryogen into the conduits 31a, 31b. The associated pressure head flushes the cryogen from both mouthpieces 31a, 31b, with the shaded area filling the space between them, as shown in Figure 3A. In Figure 3C, the plunger P is engaged with the lid 41, and the plunger P and lid 41 move together downwards (see arrow 47). Due to its size / positioning, the lid 41 engages with the left inlet orifice 37a, blocking / capping it and preventing the flow of cryogen, while in this way the right inlet orifice 37b is unobstructed. As a result, the flash of cryogen from the left mouthpiece 33a stops, but the flash from the right mouthpiece 33b continues. Note that the hatched area (flash) now only comes from the right mouthpiece 33b.

[0062] As noted above, one skilled in the art can select d and / or the downward velocity of plunger P to cause this termination of flushing from left mouthpiece 33a at a preselected time interval after the start of flushing. This time interval may be, for example, on the order of 10 to 200 milliseconds.

[0063] It should be noted that the use of a shielding mechanism 41 as shown in Figures 3A-3C is entirely optional. It is also possible to flush the sample S evenly on both sides.

[0064] As shown diagrammatically in FIG. 7, a sealing element 91 can be provided at the surface level 9 of the cryogen bath. The sealing element can be connected to the plunger P and move with it. In this way, the cryogen bath with the plunger can form a closed piston. The seal 91 can be provided, for example, in the form of a single Teflon O-ring. This O-ring (tight at LN2 temperatures) prevents spraying and splashing. The O-ring can also ensure a more controlled flow of ethane from the nozzle, which is determined solely by the ratio of the plug diameter to the nozzle opening time and the downward velocity of the plug.

[0065] As also shown in FIG. 7, a further sealing element 93 can be provided. This further sealing element 93 is provided above the first sealing element 91. The further sealing element 93 is connected to the piston P and is movable therewith. This sealing element 93 can also be an O-ring, which allows the release of liquid ethane after the downward action of the plunger P and prevents ethane from leaking when pushing downwards, especially when resetting the plug to its upward position. In the embodiment shown, the plunger comprises two further channels 38a, 38b (separated from the two conduits) connected to the channel formed by the intervening gap 35. The cryogenic fluid reservoir comprises one or more outlets 83a. At least two seal elements 91, 93 are movably connected to the plunger P, and a vertical distance exists between the two seal elements 91, 93, so that the relative position between the plunger P and the cryogenic bath determines the flow channels 81a, 81b connecting the additional channels 38a, 38b to the respective outlets 83a, 83b. This is useful, for example, when the plunger P is filled with cryogenic fluid and the plunger moves upward. When the plunger is in the position shown in FIG. 7, the cryogenic fluid present in the intervening gap 35 can be easily removed from the system. If the plunger is moved further upward, the first seal element 91 again closes the outlet 83a. If the plunger is moved further downward, the flow paths to the outlets 83a, 83b are also closed.

[0066] It should be noted that the plunger device described herein can be effectively used as a jet freezing device. One additional advantage of the plunger device described herein is that the submerged position of the plunger in the cryogenic bath allows the device described herein to also be used as a plunge freezing device. To this end, a tool can simply enter the plunger device to establish plunger freezing. In an alternative embodiment, the cryogenic bath can include a pumping device to establish forced flow through the plunger conduit, so that jet freezing can occur without actually moving the plunger into the bath.

[0067] Embodiment 2 FIG. 4 is a highly schematic illustration of an embodiment of a CPM that can be used to examine samples prepared according to the present invention; more specifically, it shows an embodiment of a transmission microscope M, in this case a TEM / STEM (in the context of the present invention, for example, it could equally effectively be an ion-based microscope). In the illustration, within a vacuum enclosure V, an electron source 2 (e.g., a Schottky emitter, etc.) generates an electron beam C that traverses an electron-optical illumination device 4, which serves to concentrate / focus them onto a selected portion of the sample S (e.g., which may be (locally) thinned / flattened). This illumination device 4 has an electron-optical axis C′ and generally comprises various electrostatic / magnetic lenses, a (scanning) deflector D, correctors (e.g., stigmators), etc., and may also typically comprise a condenser system (item 4 as a whole may sometimes be referred to as the “condenser system”).

[0068] The specimen S is held on a specimen holder H, which can be positioned with multiple degrees of freedom by a positioning system / stage A; for example, the specimen holder H may include fingers that can move (among other things) in the XY plane (referring to the depicted Cartesian coordinate system, typically movement parallel to Z and tilting about (at least) X / Y are also possible). Such movement allows different portions of the specimen S to be illuminated / imaged / inspected by the electron beam moving along axis C' (in the Z direction) (and / or a scanning motion is performed as an alternative to beam scanning). The cooling device H' is in intimate thermal contact with the specimen holder H, and the cooling device H' can be maintained at, for example, a cryogenic temperature, e.g., using a vat of cryogenic coolant to achieve and maintain the desired cryogenic temperature.

[0069] The (focused) electron beam C traveling along axis C' interacts with the sample S in such a way that various types of "stimulating" radiation emanate from the sample S, including (for example) secondary electrons, backscattered electrons, X-rays, and optical radiation (cathodoluminescence). If desired, one or more of these radiation types can be detected using an analytical device 6, e.g., a combined scintillator / photomultiplier tube or EDX (energy dispersive X-ray spectroscopy) module, in which case an image can be constructed using essentially the same principles as in an SEM. However, alternatively or supplementarily, one can consider electrons that traverse (pass through) the sample S, exit (diverge) from the sample, and continue propagating further along axis C' (albeit substantially deflected / scattered to some degree). Such a transmitted electron beam enters an imaging system (combined objective / projection lens) 8, which typically includes various electrostatic / magnetic lenses, deflectors, correctors (such as stigmators), etc. In normal (non-scanning) TEM mode, this imaging system 8 can be retracted / withdrawn, if desired, away from the axis C' (as shown diagrammatically by arrow 10') to focus the transmitted electron beam onto a phosphor screen 10. An image (or diffractogram) of (a portion of) the sample S is formed on the screen 10 by the imaging system 8, which can be viewed through a viewing port 12 located in a suitable part of the wall of the enclosure V. The retraction mechanism for the screen 10 can be, for example, mechanical / electrical and is not shown here.

[0070] As an alternative to viewing the image on the screen 10, one can instead take advantage of the fact that the depth of convergence of the electron beam emerging from the imaging system 8 is typically quite large (e.g., on the order of about one meter). As a result, various other types of analytical equipment can be used downstream of the screen 10: -TEM camera 14. In camera 14, the electron beam can be processed by a controller E to form a still image (or diffractogram) that is displayed on a display device (not shown), such as a flat panel display. When not needed, camera 14 can be retracted / withdrawn (as shown diagrammatically by arrow 14') outwardly from the direction of axis C'. - STEM imager (camera) 16. The output from imager 16 can be recorded as a function of the (X, Y) scanning position of beam C on sample S, and an image can be constructed that is a "map" of the output from imager 16 as a function of X, Y. Imager 16 can comprise a single pixel, e.g., with a diameter of 20 mm, as opposed to the matrix of pixels characteristically present in camera 14. Furthermore, imager 16 generally has a much higher acquisition rate (e.g., 106 points / second) than camera 14 (e.g., 102 images / second). Again, when not needed, imager 16 can be retracted / pulled out of the direction of axis C' (as indicated diagrammatically by arrow 16'). (In the case of a donut-shaped annular dark-field imager 16, such retraction is not necessary; however, in such an imager, for example, a central hole allows the beam to pass when the imager is not in use.) As an alternative to imaging using the camera 14 or the imaging device 16, it is also possible to activate a spectroscopic device 18, which may for example be an EELS module (EELS=Electron Energy Loss Spectroscopy).

[0071] It should be noted that the order / location of items 14, 16 and 18 is not strict and many possible variations are possible. For example, spectroscopic device 18 could be integrated into imaging system 8.

[0072] Note that a controller (computer processor) E is connected to the various components shown via control lines (bus) E'. This controller E can provide various functions, such as synchronizing operations, providing setpoints, processing signals, performing calculations, and displaying messages / information on a display device (not shown). Needless to say, the (schematically drawn) controller E can be (partially) inside or outside the enclosure V, and can have a single or multiple structure, as desired. Those skilled in the art will appreciate that the interior of the enclosure V need not be maintained at a strict vacuum; for example, in so-called "environmental TEM / STEM," a background atmosphere of a given gas is intentionally introduced / maintained within the enclosure V. Those skilled in the art will appreciate that, in practice, it may be advantageous to limit the volume of the enclosure V to closely surround the axis C', so that it expands to accommodate structures such as the source 2, sample holder H, screen 10, camera 14, imaging device 16, and spectrometer 18.

[0073] The sample S shown in Fig. 4 may be, for example, a sample that has been subjected to a vitrification procedure according to the invention. Thanks to the cooling device H', such a sample can be kept at cryogenic temperatures while in the CPMM (and while being transported / stored). For this purpose, for example, the following embodiment can be adopted: The cooling device H' is thermally connected intimately (eg via copper rods and / or blades) to the holder H and comprises a dewar / flask which can be filled with a cryogen. The composite structure H+H′ can be inserted / removed from the CPMM and thereby seated / clamped to the receiver part of the positioning system A.

[0074] See, for example, the arrangements described in US Patent Application No. US2012 / 0112064A1 and similar arrangements known per se to those skilled in the art.

[0075] Further embodiments 5A-5D, embodiments of a nozzle opening as defined herein are described.

[0076] FIG. 5A illustrates one embodiment of a mouthpiece 39a, comprising a nozzle plate 101 with a single central nozzle opening 111 and a total of six peripheral nozzle openings 121-126 evenly distributed around the central nozzle opening 111. Note that in this embodiment, all nozzle openings 111, 121-126 have substantially equal diameters. Additionally, this embodiment, with a total of seven nozzle openings of substantially equal size, most effectively utilizes the total area of ​​the mouthpiece 39a to evenly distribute cooling power across the surface of the sample, resulting in the peripheral portions of the sample carrier being cooled at least simultaneously with the central portion of the sample carrier. This improves sample vitrification compared to known techniques.

[0077] 5B shows one embodiment of a mouthpiece 39a, which includes a nozzle plate 201 with two nozzle openings, a central nozzle opening 211, and a single peripheral nozzle opening 221. The nozzle plate 201 also has a central portion 203 and a peripheral portion 202, which are connected by mechanical studs 231-234 to allow the central nozzle plate 203 to maintain its position. The studs 231-234 effectively subdivide the single peripheral nozzle into four sub-openings 221a-221d, even though there is actually only a single opening.

[0078] 5C shows one embodiment of a mouthpiece 39a having a single central nozzle opening 311 and a total of five peripheral nozzles provided evenly distributed around the central nozzle opening 311, for a total of six nozzle openings 311, 321-325. The peripheral nozzle openings 321-325 are larger in size compared to the central nozzle 311. In this way, the peripheral portion of the sample carrier is cooled at least simultaneously with the central portion of the sample carrier.

[0079] Note that, as defined herein, the central and peripheral portions of the sample carrier may be defined as follows: the central portion corresponds to the innermost portion, which corresponds to approximately 1 / 3 of the total radius of the circle, and the peripheral portion corresponds to the outermost portion, which corresponds to approximately 2 / 3 of the total radius of the circle. This can be seen most clearly in FIG. 5A, where the central nozzle opening 111 (having an arbitrary radius r) occupies the central portion of the mouthpiece 101, and the peripheral nozzle openings 121-126 (also having the same arbitrary radius r) occupy the peripheral portion of the mouthpiece 101. Thus, the total radius of the mouthpiece 101 corresponds to approximately three times the arbitrary radius r (one time the radius of the central nozzle opening 111 and two times the radius of the peripheral nozzle opening 121). In embodiments, the central portion may be 20% larger or smaller, meaning that the central radius may be 20% larger or smaller.

[0080] 5D shows an embodiment with a total of five nozzle openings 411, 421-424, where the central nozzle opening 411 is circular and the peripheral nozzle openings 421-424 are elliptical, so here the central nozzle has a different shape compared to the peripheral nozzles.

[0081] 5E shows one embodiment of mouthpiece 39a with nozzle plate 501 that includes a total of three oval nozzles 511-513. Thus, in this embodiment, there is no central nozzle opening.

[0082] 5F shows another embodiment in which there is no central nozzle opening, where a total of two nozzle openings 611, 612 are provided in the nozzle plate 601 to define a mouthpiece 39a having at least two nozzle openings 611, 612.

[0083] With reference to the embodiments shown in Figures 5A-5F, it should be noted that the at least two nozzle openings may be embodied in several different ways. The at least two nozzles may be embodied according to one or more of the embodiments shown in Figures 5A-5F, or a combination thereof. In particular, There may be a total of two or more nozzles. There may be up to seven nozzles in total, although additional nozzles are also contemplated. There may be at least one central nozzle, but one or more additional central nozzles are also conceivable. There may be more than one peripheral nozzle, but in particular up to six or more peripheral nozzles are also conceivable. The nozzles may have corresponding or different shapes, but as a rule corresponding shapes provide more predictable results. The nozzle may in principle have any suitable geometric shape, but a round nozzle provides more predictable results.

[0084] As described herein, the other of the two mouthpieces may also include two nozzle openings. These at least two additional nozzles may be embodied according to one or more of the embodiments shown in Figures 5A-5F, or a combination thereof. The first mouthpiece may be exactly or substantially identical to the second mouthpiece. Alternatively, the first mouthpiece may be embodied differently from the second mouthpiece.

[0085] The desired protection is provided by the appended claims.

Claims

1. 1. A method of preparing a sample of a cryogenic fluid, the sample being subjected to rapid cooling using a cryogenic fluid, the method comprising: - providing a sample on a planar sample carrier; - providing two conduits for transporting a cryogenic fluid, each of the two conduits having a flared end and arranged with one end of the two conduits facing each other with a gap between them; - placing the sample carrier with the sample in the gap; - pumping or piston-acting cryogenic fluid into the conduits from each of the one ends of the conduits so that cryogenic fluid is simultaneously flushed from each of the other ends of the conduits, thereby suddenly immersing the sample in cryogenic fluid from two opposite sides; The method further comprising providing a nozzle plate at least on one of said ends of said conduit, said nozzle plate having at least two nozzle openings.

2. The method of claim 1 , wherein the nozzle plate comprises a central nozzle disposed in a central portion of the nozzle plate and at least one peripheral nozzle disposed around the central nozzle.

3. The method of claim 2 , wherein the central nozzle and the peripheral nozzle are the same size.

4. A method as described in claim 2 or 3, wherein the nozzle plate has at least six peripheral nozzles evenly arranged around the central nozzle.

5. The method according to any one of claims 1 to 4, wherein the at least two nozzle openings have the same shape and are arranged symmetrically with respect to a central axis of the nozzle plate.

6. The method of claim 2 , wherein the central nozzle has a smaller size compared to the at least one peripheral nozzle.

7. The method according to any one of claims 1 to 6, further comprising providing a nozzle plate at the other of said one end of said conduit, said nozzle plate comprising said at least two nozzle openings.

8. - the conduit is arranged in a plunger, the other end of the conduit having an inlet opening on the underside of the plunger, the gap being provided as a slot on the upper side of the plunger; - a reservoir of cryogenic fluid is provided below the plunger, 8. The method of any one of claims 1 to 7, wherein the sample is inserted into the slot using a tool that applies downward pressure to the plunger, thereby at least partially immersing the plunger in the cryogenic fluid in the vessel and forcing the cryogenic fluid in the vessel to enter the inlet opening and exit via the one end of the conduit.

9. The method described in claim 8, wherein a shield closing the other end of one of the two conduits is connected to the other end so that the flushing from both of the two conduits starts simultaneously, but the flushing from one of the two conduits is terminated after a given time interval.

10. - the plunger is surrounded by at least one sealing element, which may be provided at the surface level of the cryogenic fluid in the vessel and which is connected to the plunger and is capable of moving together with the plunger; the plunger is surrounded by at least one further sealing element, the further sealing element being positioned at a distance from the at least one sealing element and out of contact with the cryogenic fluid in the vessel, the further sealing element being connected to the plunger and being able to move together with the plunger; a channel is formed between the at least one sealing element and the at least one further sealing element; a further channel in the plunger, the further channel being capable of communicating the gap with the channel and being separated from the two conduits; 10. The method according to claim 8 or 9, wherein the plunger is immersed in the cryogenic fluid in the bath before the cryogenic fluid is removed from the gap via the channel and the further channel.

11. 1. An apparatus for preparing a sample of a cryogenic fluid, whereby said sample is subjected to rapid cooling using a cryogenic fluid, said apparatus comprising: a pair of conduits for transporting a cryogenic fluid, each end of the pair of conduits being flared toward a tip, the ends of the conduits being arranged opposite each other with a gap therebetween, the gap being provided with a planar sample carrier in which a sample can be received; a flushing mechanism for pumping or piston-acting cryogenic fluid from each of the one ends of the conduits so that cryogenic fluid is simultaneously flushed from each of the other ends of the conduits, immersing the sample in cryogenic fluid from two opposite sides; The apparatus includes a nozzle plate at at least one of said one ends of said conduit, said nozzle plate including at least two nozzle openings.

12. The apparatus of claim 11, having a nozzle plate configured to be used in a method according to any one of claims 2 to 7.

13. - the conduit is arranged in a plunger, the other end of the conduit having an inlet opening on the underside of the plunger, the gap being provided as a slot on the upper side of the plunger; - a reservoir of cryogenic fluid is provided below the plunger, 13. The apparatus of claim 11 or 12, wherein the sample is inserted into the slot using a tool that applies downward pressure to the plunger, thereby at least partially immersing the plunger in the cryogenic fluid in the vessel and forcing the cryogenic fluid in the vessel to flow into the inlet opening and out through the one end of the conduit.

14. The device of claim 13, having a plunger configured for use in a method according to any one of claims 8 to 10.

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