Method of time-resolved charged-particle microscopy

Abstract

To provide an improved method of time-resolved charged particle microscopy.SOLUTION: The invention relates to a method of time-resolved charged particle microscopy, comprising providing a sample for charged particle microscopy, wherein the sample comprises first particles and second particles, and wherein the sample comprises a barrier material between the first particles and the second particles. The method further includes liquefying at least a portion of the barrier material to enable interaction between the first particles and the second particles. Finally, the resulting interaction between the first and second particles can be observed with a charged particle microscope.SELECTED DRAWING: Figure 14b

Description

[Technical Field]

[0001] The present disclosure relates to a method for time-resolved charged particle microscopy. [Background technology]

[0002] Charged particle microscopy is a well-known and increasingly important technique for imaging microscopic objects, particularly in the form of electron microscopy. Over time, the basic type of electron microscope has evolved into a number of well-known instrument families, such as transmission electron microscopes (TEMs), scanning electron microscopes (SEMs), and scanning transmission electron microscopes (STEMs), as well as various variants, such as so-called "dual beam" instruments (e.g., FIB-SEMs) that additionally use a "machining" focused ion beam (FIB) to enable supporting functions such as ion beam milling or ion beam induced deposition (IBID). More specifically, In an SEM, irradiation of a sample by a scanning electron beam causes the sample to emit "auxiliary" radiation, e.g., in the form of secondary electrons, backscattered electrons, X-rays, and photoluminescence (infrared, visible, and / or ultraviolet photons), and one or more components of this emitted radiation flux are then detected and used to build up an image. In a -TEM, the electron beam used to irradiate the sample is chosen to be of sufficiently high energy to penetrate the sample (for this purpose, the sample is generally thinner than in an SEM sample), and the transmitted electron flux emitted from the sample can then be used to create an image. When such a TEM is operated in scanning mode (thus becoming a STEM), its image is accumulated during the scanning motion of the irradiating electron beam.

[0003] It is desirable to study (bio)chemical experiments in greater detail, both in terms of spatial and temporal detail. The ability to study (bio)chemical experiments in a charged particle microscope makes it possible to see more spatial details of the experiment. However, the time constants of (bio)chemical reactions are very low, which imposes constraints on sample preparation.

[0004] Prior art documents, such as EP 1 803 141 A1, use microreactors to observe particles or portions thereof in a fluid. These microreactors include two cover layers held at a small distance from each other by a spacer. The two cover layers thus define a channel, which is provided with an inlet and an outlet. This allows fluid to be introduced into the channel. Each cover layer exhibits a number of recesses that are thin enough to be transparent to the electrons used in TEM. These recesses are positioned so that an electron beam impinging perpendicularly on the cover layer and passing through one recess also passes through the corresponding recess in the other cover layer. This allows the fluid between the two corresponding recesses, and any particles and / or cells within the fluid, to be observed by TEM.

[0005] Another microreactor device is known from EP 2 316 565 A1 in the name of the applicant.

[0006] These microreactors are technically complex and have the drawback that not all parts of the microreactor are transparent to electrons, which leads to the fact that studying (bio)chemical reactions in a time-resolved manner using charged particle microscopy is very time-consuming and difficult. Summary of the Invention

[0007] It is therefore an object of the present invention to provide a method for time-resolved charged particle microscopy that overcomes at least one of the drawbacks of the prior art.

[0008] To this end, the present disclosure provides a method of time-resolved charged particle microscopy as set forth in claim 1.

[0009] A method as defined herein includes providing a sample for charged particle microscopy, the sample including a first particle and a second particle, and the sample including a barrier material between the first particle and the second particle.

[0010] As defined herein, the method includes liquefying at least a portion of a barrier material to allow interaction between the first and second particles. By using a barrier material such as vitreous ice, the first and second particles can be kept separated from each other until a desired time. Thus, the barrier material can be configured to prevent the first and second particles from interacting with each other.

[0011] The barrier material defined herein is configured to be liquefiable, meaning that it can be brought into a fluid-like state to at least partially remove the barrier between the first particle and the second particle. By liquefying the barrier material, the first particle and the second particle can come into contact with each other. This can be due to Brownian motion of the particles, which leads to particle diffusion, so that the first particle and the second particle can encounter each other. The liquefied barrier material can provide a medium in which the first particle and the second particle exhibit Brownian motion. In other words, the liquefied barrier material can act as a solvent for the first particle and the second particle. Then, when the particles encounter each other, they can interact with each other. The resulting interaction between the first particle and the second particle, which can be a chemical reaction or a protein-protein interaction, can then be observed in a charged particle microscope, such as a (cryogenic) transmission electron microscope.

[0012] This provides an improved method for observing interactions between particles. The provided method does not require the use of expensive and complex mini-reactors. In contrast, it uses a liquefiable barrier material that can be relatively inexpensive and can be applied to standard grids used in charged particle microscopy. Indeed, an improved method for time-resolved charged particle microscopy is provided, thereby achieving the objectives defined herein.

[0013] Advantageous embodiments are described below.

[0014] In one embodiment, liquefying the barrier material includes heating the barrier material. Heating the barrier material can be performed with great spatial and temporal precision. By way of example, the heat may be applied locally, resulting in localized interactions between the first particles and the second particles. The heat may be applied at different time intervals and for different durations.

[0015] Heat may be applied to the sample for charged particle microscopy, and there are various ways to liquefy the barrier material.

[0016] In one embodiment, a laser is used to liquefy the barrier material. Lasers can apply heat very locally, resulting in very localized interactions between the first and second particles. Lasers are also well-suited to applying heat in a very controlled manner.

[0017] In one embodiment, a charged particle beam of a charged particle microscope is used to liquefy the barrier material. The charged particle beam may be a focused ion beam that allows removing at least a portion of the barrier material between the first particle and the second particle, thereby allowing the first particle and the second particle to interact with each other.

[0018] In a further embodiment, the sample contains heat absorbing particles, such as gold particles, which are capable of absorbing heat and thereby liquefying the barrier material surrounding the gold particles.

[0019] In one embodiment, a charged particle beam can be used to heat the heat absorbing particles, which then liquefies the barrier material. The charged particle beam can be an electron beam used to irradiate a portion of the sample. In this way, the barrier material can be liquefied immediately before and during observation of the interaction between the first particle and the second particle.

[0020] In one embodiment, the method includes a step of re-solidifying the barrier material after the step of liquefying the barrier material. Liquefying and re-solidifying the barrier material allows for more time to observe the interaction between the first particle and the second particle. Similar to stop-motion photography, this allows different parts of the interaction to be frozen in time, which allows for a more detailed observation of the entire interaction.

[0021] Re-solidifying the material may include cooling the barrier material. If the barrier material is vitreous ice, liquefying may include locally heating the sample. The surrounding portion of the heated material remains cool. Heat from the heated portion of the sample then flows to the cooler portion surrounding the heated portion, causing the heated portion to cool again. Cooling can result in re-vitrification of the heated portion of the sample, thus causing it to re-solidify again. In this state, observations of the interaction between the first particle and the second particle can be performed.

[0022] In one embodiment, the method includes re-liquefying at least a portion of the barrier material after the re-solidifying step, and then observing the resulting interaction between the first particles and the second particles.

[0023] In one embodiment, time-resolved charged particle microscopy includes liquefying a barrier material at a first location, solidifying the barrier material at the first location, and observing interactions between first particles and second particles at the first location. To obtain further time-resolved data, the method may include liquefying the barrier material at a second location, solidifying the barrier material at the second location, and observing interactions between the first particles and second particles at the second location. In other words, the sample includes a plurality of first particles (or a plurality of particles of a first type) and a plurality of second particles (i.e., a plurality of particles of a second type). The first type particles and the second type particles can react with each other. Because there are a plurality of particles of a first type and a plurality of particles of a second type, there are many possibilities for interactions between the first type particles and the second type particles. Liquefying the barrier material at the first location allows the first type particles to interact with the second type particles, and the resulting interactions can be observed. Then, by liquefying the barrier material at the second location, further particles of the first type can interact with further particles of the second type. Thus, even if different particles are present at different locations, the method still allows the same interactions between particles of the first type and particles of the second type to be studied.

[0024] In one embodiment, the liquefaction of the barrier material at the second location is selected to have a longer duration than the liquefaction of the barrier material at the first location, which allows the resulting interaction to be observed at different time intervals, which is relatively easy to do at different locations compared to observing the interaction at a single location.

[0025] The above embodiments relate to a method of time-resolved charged particle microscopy, the method comprising: providing a sample for charged particle microscopy, the sample comprising a plurality of particles of a first type and a plurality of particles of a second type, the sample comprising a barrier material configured to substantially separate the first type of particles and the second type of particles; - liquefying at least a portion of the barrier material at the first location to allow interaction between at least one of the plurality of particles of the first type and at least one of the plurality of particles of the second type; and - observing with a charged particle microscope the resulting interaction between at least one of the plurality of particles of the first type and at least one of the plurality of particles of the second type.

[0026] A further step of the method may include liquefying at least a portion of the barrier material at the second location to allow interaction between at least one further particle of the plurality of particles of the first type and at least one further particle of the plurality of particles of the second type.

[0027] Advantageously, this allows time-resolved charged particle microscopy to be performed on multiple different parts of the sample.

[0028] This method can be carried out using standard electron microscope sample grids known to those skilled in the art. These grids contain so-called grid squares, which are subparts of the grid itself. Particles of a first type and particles of a second type may be provided on such a grid, with a barrier material keeping the particles separated from each other. The barrier material on different parts of the grid may be liquefied for different time values. For example, the barrier material at a first position (e.g., grid square 1) may be liquefied for a given time t (which may be on the order of milliseconds, depending on the expected time scale of interactions between particles) and then solidify again. At a second position (e.g., grid square 2), the barrier material may be liquefied for a longer duration, e.g., 2 *At a third location (e.g., grid square 3), the barrier material is liquefied for an even longer duration, e.g., 3 * It is liquefied for t, and the number of n positions is n * Similarly for t. This allows the resulting interactions between particles to be studied on a single grid at n discrete time intervals.

[0029] Alternatively, different time intervals can be studied per sample carrier. For example, the first grid is 1 * Use a liquefaction time of t, and the second grid is 2 * With a liquefaction time of t, the nth grid is n * Use liquefaction time t.

[0030] As described herein, at least one of the first particle and the second particle is selected from the group consisting of a nanoparticle, a biomolecule, a virus, a protein, a ligand, and / or a pharmaceutical molecule.

[0031] In one embodiment, the sample is a cryo-electron microscope sample and the barrier material comprises amorphous ice.

[0032] In one embodiment, the step of liquefying the barrier material is performed when a sample for charged particle microscopy is placed inside the charged particle microscope. The step of liquefying the barrier material is performed while the sample is inside the charged particle microscope. In other words, the sample can remain inside the charged particle microscope during the liquefaction step, so that the resulting interaction can be immediately observed. Thus, the method can be performed entirely inside the charged particle microscope.

[0033] In an alternative embodiment, the step of liquefying the barrier material is performed when the sample for charged particle microscopy is outside the charged particle microscope, thus allowing the sample to be prepared after observation of the resulting interactions.

[0034] According to one aspect, a method of preparing a sample for time-resolved charged particle microscopy is provided. The method includes providing a sample carrier for charged particle microscopy. The method further includes depositing first particles, second particles, and a barrier material on the sample carrier. The first particles, second particles, and barrier material are deposited in an order such that the first particles are provided at a distance from the second particles, and the barrier material is provided between the first particles and the second particles.

[0035] The sample preparation methods described above can be used to provide a method of preparing a sample for time-resolved charged particle microscopy that allows time-resolved charged particle microscopy to be performed according to the methods described herein.

[0036] The order of deposition and variations thereof according to the above definition will be apparent to those skilled in the art. As an example, the first particles can be deposited first, and then the barrier material can be deposited on the first particles. Then, the second particles can be deposited on the barrier material. Thus, in this embodiment, the first particles are deposited on the sample carrier before the step of depositing the second material. Furthermore, the barrier material is deposited on the sample carrier before the step of depositing the second particles. By using this order, the first particles and the second particles are separated from each other by the barrier material. The barrier material is provided between the first particles and the second particles, and the first particles are provided at a distance from the second particles. Therefore, the above order of deposition results in the ordering of the first particles, the second particles, and the barrier material as defined herein.

[0037] The above deposition sequences are merely examples, and further embodiments may be envisioned and derived by those skilled in the art. It should be noted that further non-limiting examples of different deposition sequences according to the above definitions are described in more detail herein in the description of the accompanying drawings. [Brief explanation of the drawings]

[0038] For a more complete understanding of the present disclosure and its advantages, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which: [Figure 1] 1 shows a longitudinal section of a charged particle microscope, in particular a transmission charged particle microscope. [Figure 2] 1 shows a first embodiment of a sample for use in the time-resolved charged particle microscopy described herein. [Figure 3] 1 shows a second embodiment of a sample for use in the time-resolved charged particle microscopy described herein. [Figure 4a] 1 illustrates intermediate steps for preparing a sample for use in the time-resolved charged particle microscopy method described herein. [Figure 4b] 10A and 10B show third and fourth embodiments, respectively, of a sample for use in the time-resolved charged particle microscopy method described herein. [Figure 4c] 10A and 10B show third and fourth embodiments, respectively, of a sample for use in the time-resolved charged particle microscopy method described herein. [Figure 5] 10 illustrates a fifth embodiment of a sample for use in the time-resolved charged particle microscopy methods described herein. [Figure 6] 10 illustrates a sixth embodiment of a sample for use in the time-resolved charged particle microscopy methods described herein. [Figure 7] 10 shows a seventh embodiment of a sample for use in the time-resolved charged particle microscopy methods described herein. [Figure 8] 10 illustrates an eighth embodiment of a sample for use in the time-resolved charged particle microscopy methods described herein. [Figure 9] 10 illustrates a ninth embodiment of a sample for use in the time-resolved charged particle microscopy methods described herein. [Figure 10] 10 illustrates a tenth embodiment of a sample for use in the time-resolved charged particle microscopy methods described herein. [Figure 11] 11 illustrates an eleventh embodiment of a sample for use in the time-resolved charged particle microscopy methods described herein. [Figure 12] 12 illustrates a twelfth embodiment of a sample for use in the time-resolved charged particle microscopy methods described herein. [Figure 13]13 illustrates a thirteenth embodiment of a sample for use in the time-resolved charged particle microscopy methods described herein. [Figure 14a] 1 illustrates different time points of one embodiment of time-resolved charged particle microscopy as disclosed herein. [Figure 14b] 1A-1C illustrate different time points of one embodiment of time-resolved charged particle microscopy as disclosed herein. [Figure 14c] 1A-1C illustrate different time points of one embodiment of time-resolved charged particle microscopy as disclosed herein. [Figure 14d] 1 illustrates different time points of one embodiment of time-resolved charged particle microscopy as disclosed herein. [Figure 15] 1 illustrates a further embodiment of a sample for use in the time-resolved charged particle microscopy described herein. DETAILED DESCRIPTION OF THE INVENTION

[0039] Figure 1 (not to scale) is a highly schematic illustration of a charged particle microscope M that can be used in the methods disclosed herein. More specifically, one embodiment of a transmission microscope M, in this case a TEM / STEM, is shown. In Figure 1, within a vacuum enclosure 102, an electron source 104 generates an electron beam B propagating along an electron-optical axis B' and traversing an electron-optical illuminator 106, which serves to direct / focus the electrons onto a selected portion of a sample S (which can, for example, be (locally) thinned / flattened). Also shown is a deflector 108 that can be used to (among other things) effect a scanning motion of the beam B.

[0040] The sample S is held on a sample holder H, which can be positioned with multiple degrees of freedom by a positioning device / stage A, which moves a cradle A' to which the holder H is (removably) fixed. For example, the sample holder H may be equipped with fingers that can move (among other things) in the XY plane (see the depicted Cartesian coordinate system). Typically, movement parallel to Z and tilting about X / Y are also possible). Such movement allows different portions of the sample S to be illuminated / imaged / inspected by the electron beam B traveling along axis B' (Z direction) (and / or allows a scanning movement to be performed as an alternative to beam scanning). If desired, an optional cooling device (not depicted) can be in intimate thermal contact with the sample holder H, thereby maintaining the sample holder H (and the sample S thereon) at, for example, cryogenic temperatures.

[0041] The electron beam B interacts with the sample S, causing various types of "stimulated" radiation to be emitted 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 analysis device 122, which may incorporate, for example, a scintillator / photomultiplier tube or an EDX (energy dispersive X-ray spectroscopy) module. In such cases, images can be constructed using essentially the same principles as in an SEM. However, electrons that traverse (pass) the sample S and continue to propagate along axis B' (effectively, typically deflected / scattered to some extent) can be alternatively or supplementarily investigated. Such a transmitted electron flux enters a projection system (projection lens 124), which typically includes various electrostatic / magnetic lenses, deflectors, correctors (such as stigmators), etc. In normal (non-scanning) TEM mode, this projection system 124 can focus the transmitted electron beam onto a phosphor screen 126, which can be retracted / retracted, if necessary, out of axis B' (as indicated diagrammatically by arrow 126'). An image (or diffractogram) of (a part of) the sample S is formed by the projection system 124 on the screen 126, which can be viewed through an observation port 128 located in a suitable part of the wall of the enclosure 102. The retraction mechanism for the screen 126 can, for example, be mechanical and / or electrical in nature and is not shown here.

[0042] Instead of viewing an image on a screen 126, one can take advantage of the fact that the depth of focus of the electron beam leaving the projection system 124 is typically very large (e.g., on the order of one meter). Therefore, downstream of the screen 126, various other types of analytical devices can be used, such as: -TEM detector (camera) 130. In camera 130, the electron beam can be processed by controller / processor 120 to form a still image (or diffractogram) that can be displayed on a display device (not shown), such as a flat panel display. When not needed, camera 130 can be retracted / retracted (as indicated diagrammatically by arrow 130') so that it is out of the way of axis B'. - STEM detector (camera) 132. The output from camera 132 can be recorded as a function of the (X,Y) scanning position of beam B on sample S, and an image can be constructed that is a "map" of the output from camera 132 as a function of X,Y. Camera 132 can include a single pixel, e.g., having a diameter of 20 mm, as opposed to a matrix of pixels that is typically present in camera 130. Furthermore, camera 132 can typically capture images at a rate of 100x1000 per second (e.g., 10x1000 per second). 2 images) at a much higher acquisition rate (e.g., 10 per second) 6 Again, when not needed, the camera 132 can be retracted / retracted (as indicated diagrammatically by arrow 132') out of the way of axis B' (however, such retraction is not necessary in the case of, for example, a donut-shaped annular dark field camera 132; in such cameras, a central hole allows the passage of the light beam when the camera is not in use). As an alternative to imaging using the camera 130 or 132, a spectroscopic detector 134 can also be activated, which can be for example an EELS module.

[0043] It should be noted that the order / position of items 130, 132 and 134 is not strict and many possible variations are possible. For example, spectroscopic detector 134 could be integrated into projection system 124.

[0044] In the illustrated embodiment, the microscope M further comprises a retractable X-ray computed tomography (CT) module generally designated by the reference numeral 140. In computed tomography (also called tomography), a source and a detector (opposing each other) are used to interrogate a sample along different lines of sight to obtain transmitted views of the sample from various viewpoints.

[0045] It should be noted that the detectors 130, 132, and 134 are part of an imaging system (generally designated by reference numeral 200). The imaging system is configured to generate image signals based on information from the charged particle detectors 130, 132, and 134 and may be part of the detectors or may be separate from the detectors. A controller (computer processor) 120 is connected to the various illustrated components via control lines (bus) 120′. This controller 120 may provide various functions such as synchronizing operations, providing set points, processing signals, performing calculations, and displaying messages / information on a display device (not shown). Of course, the (schematically illustrated) controller 120 may be (partially) inside or outside the enclosure 102 and may have a single or composite structure as desired.

[0046] Those skilled in the art will understand that it is not necessary to maintain a strict vacuum inside the enclosure 102. For example, in so-called "environmental TEM / STEM," a background atmosphere of a given gas is intentionally introduced / maintained inside the enclosure 102. Those skilled in the art will also understand that, although in practice this will take the form of a small tube (e.g., on the order of 1 cm in diameter) through which the electron beam used passes, where possible, it may be advantageous to limit the volume of the enclosure 102 to essentially encompass the axis B' extending to accommodate structures such as the source 104, the sample holder H, the screen 126, the cameras 130 and 132, the spectroscopic detector 34, etc.

[0047] Thus, the charged particle microscope M shown in FIG. 1 comprises a charged particle optical column O for directing a charged particle beam B onto a sample, a sample holder H for holding a sample S, and charged particle detectors 122, 126, 130, 132, 134 having an imaging system 200 for generating image signals based on information from the charged particle detectors.

[0048] The charged particle microscope M shown in Figure 1 can be configured for time-resolved charged particle microscopy. The charged particle microscope M shown in Figure 1 can be configured for so-called cryo-electron microscopy, in which a sample is studied at cryogenic conditions. Those skilled in the art will be familiar with both of these aspects of charged particle microscopy.

[0049] Referring first to FIG. 2 , a first embodiment of a sample 1 for use in time-resolved charged particle microscopy is shown. The sample 1 includes a first particle 21 and a second particle 22. The first particle 21 and the second particle 22 are selected as desired research subjects for studying biological and / or chemical interactions between the two particles. The particles 21 and 22 are separated from each other. In the illustrated embodiment, the separation is established by two intermediate layers 31 and 32. The first intermediate layer 31 encapsulates the first particle 21. The second intermediate layer 32 encapsulates the second particle 22. The two intermediate layers 31 and 32 separate the first particle and the second particle from each other. Thus, the intermediate layers 31 and 32 form a barrier material extending between the first particle and the second particle. In this embodiment, the barrier material is such that the first particles 21 are spaced apart from the second particles 22 and the barrier materials 31, 32 are at least partially disposed between the first particles 21 and the second particles 22.

[0050] The barrier material 31 ensures that the first particles 21 and the second particles 22 cannot reach each other, thus preventing interactions between the first particles 21 and the second particles 22 from occurring.

[0051] It should be noted in Figure 2 that each intermediate layer 31, 32 forms a separate barrier material 31, 32, respectively. However, it is also possible that only one of the layers 31, 32 effectively forms a barrier material.

[0052] 14a-14d, an embodiment of time-resolved charged particle microscopy will now be described, using sample 1 as described above with reference to FIG.

[0053] FIG. 14a shows the state of sample 1a before the experiment (i.e., similar to the sample described above with respect to FIG. 2), with first particle 21, second particle 22, and barrier material 31, 32 (here formed by two separate intermediate layers 31, 32) located between first particle 21 and second particle 22. The barrier material 31, 32 is configured to keep first particle 21 and second particle 22 at a distance from each other. This can be achieved, for example, by having a solid barrier material. In one embodiment, the solid barrier material can be vitreous ice, which is advantageous for use in cryo-EM experiments.

[0054] FIG. 14a shows a relatively static initial state, which can be maintained for a desired amount of time.

[0055] 14b shows sample 1b during the first step of the experiment, where barrier material 31 is liquefied to obtain liquefied barrier material 33. Because the barrier properties of liquefied barrier material 33 are significantly reduced compared to barrier material 31, particles 21, 22 can move through liquefied barrier material 33, for example by Brownian motion. The reduced barrier properties allow first particle 21 and second particle 22 to interact with each other, which in the illustrated embodiment results in particle complex 23 (first particle 21 is connected to second particle 22, dashed lines indicate the original positions of first particle 21 and second particle 22).

[0056] Obtaining a liquefied barrier material 33 may in one embodiment be done by applying heat to the barrier material 31. Heating the barrier material 31 makes it more fluid or liquid, thereby reducing the barrier properties of the barrier material 31. The application of heat may be done very locally, so that the unheated parts of the sample remain in a static initial state (as shown in Figure 14a).

[0057] In this regard, it should be noted that the samples shown throughout the figures only show a single first particle 21 and a single second particle 22. However, it will be apparent to those skilled in the art that most samples will contain large amounts of these particles.

[0058] The sample 1b shown in Figure 14b can be observed with a charged particle microscope M. This allows particle complexes 23 formed by the interaction between first particles 21 and second particles 22 to be observed with, for example, an electron microscope.

[0059] To enhance the observation capabilities of sample 1b, the liquefied barrier material 33 can be re-solidified. This is shown in FIG. 14c. Here, the liquefied barrier material 33 shown in FIG. 14b is re-solidified, thereby at least partially restoring the barrier properties of barrier material 31. The barrier properties of barrier material 31 initially ensured that first particle 21 and second particle 22 were maintained at a distance from each other. In the embodiment shown in FIG. 14c, the restored barrier properties of re-solidified barrier material 34 ensure that particle complexes 23 are preserved, so that they can be easily studied with a charged particle microscope M.

[0060] Re-solidification of the liquefied barrier material 33 may, in one embodiment, be achieved by cooling the liquefied barrier material 33. Cooling is relatively easy to apply, especially to locally heated barrier materials 31. The material surrounding the sample 1 may be used to cool the locally heated barrier material 31.

[0061] In one embodiment, the sample is a cryogenic EM sample at cryogenic temperatures (i.e., below -150 degrees Celsius). The sample is locally heated to liquefy the barrier material at specific locations (i.e., vitrified ice by reheating above 0°C), while the surrounding portion of the sample remains cold. Residual heat can be conducted to the surrounding cold portion of the sample, which results in the re-solidification of the previously melted vitreous ice.

[0062] To observe different points in time of a given interaction between a first particle 21 and a second particle 22, the steps shown in Figures 14a-14c may be repeated at least once.

[0063] Figure 14d shows the situation where the re-solidified barrier material 34 of Figure 14c is re-liquefied, resulting in a liquefied barrier material 33. The (intermediate) particle complex 23 formed by the interaction of the first particle 21 with the second particle 23 can complete the transformation process to form the final particle complex 24. This final particle 24 can be observed under a charged particle microscope M in the state shown in Figure 14d, or the liquefied barrier material 33 can be re-solidified again (not shown).

[0064] Thus, the method described herein allows for time-resolved charged particle microscopy to be performed on the interaction of a first particle 21 with a second particle 22, allowing for the observation and imaging of intermediate particles 23 and final particles 24. It will be appreciated that the steps of liquefying the barrier material (by applying heat locally) and re-solidifying the barrier material (e.g., by cooling through thermal conduction of residual heat to the sample surroundings) can be performed multiple times in a very controlled manner to provide a relatively small time step between each observation, thereby providing time-resolved charged particle microscopy with a relatively high time resolution.

[0065] Examples of samples for use in the methods described in Figures 14a-14d, and how to prepare them, are discussed in Figures 2-13.

[0066] As mentioned above, Figure 2 shows a first embodiment of a sample 1 for use in time-resolved charged particle microscopy. The sample 1 includes a first particle 21 and a second particle 22, separated from each other by two intermediate layers 31, 32. These intermediate layers 31, 32 act as a barrier extending between the first particle 21 and the second particle 22.

[0067] This embodiment can be prepared by providing a sample carrier 11, then first forming a layer of a first intermediate layer 31 containing first particles 21, and then forming a layer of a second intermediate layer 32 containing second particles 22 on the first intermediate layer 31. The first intermediate layer 31 may be formed by providing a liquid layer containing the first particles 21 and applying the solution to the sample carrier 11. After applying the solution, the layer can be solidified, for example, by vitrifying the layer (a technique known from preparing biological samples in cryo-EM). After solidifying the first layer 31, a second layer 32 containing second particles 22 can be applied on top of the first layer 31. After application, the second layer 32 can be solidified in a similar manner, for example, by vitrifying the second layer 32.

[0068] Other techniques for preparing multilayer samples such as those shown in Figure 2 can be used as well. For example, the first layer 31, including the first particles 21, such as nanoparticles, can be a relatively viscous liquid at room temperature, including a hardener, so that the layer substantially solidifies after application to the grid 11. The same can be true for the second layer 31, including the second particles 22. During the experiment, the solidified layer can be liquefied, such as by heat or a solvent. This technique is particularly suitable for nanoparticles.

[0069] 3 shows a second embodiment of Sample 1 that can be used in the methods described herein. Again, Sample 11 includes first particles 21, second particles 22, and a barrier material 31 disposed between first particles 21 and second particles 22.

[0070] Figures 4a and 4b show steps for preparing the sample of Figure 3. In Figure 4a, a sample carrier 11 is provided and first and second particles 21, 22 are deposited on the surface of the sample carrier 11. Due to the fact that the particles 21, 22 are deposited on the surface of the sample carrier, they are unlikely to interact with each other (the movement of the particles 21, 22 is restricted). To allow movement, for example by Brownian movement, a medium formed by a liquefied barrier material 31 is required. The liquefied barrier material 31 allows Brownian movement of the first particles 21 and the second particles 22 within the medium 31.

[0071] The barrier material 31 can be applied by depositing a liquid onto the sample carrier 11 on which the first and second particles have been deposited. The liquid can then be solidified, for example by cooling the liquid to a temperature of -150°C or lower for biological samples, so as to reach the static initial state referred to in Figure 14a. In this way, the state shown in Figure 4b can be reached.

[0072] In another embodiment shown in Figure 4c, the barrier material 31 is deposited in the form of large solid pillars 31 on top of the sample carrier 11. If the number of particles 21, 22 deposited on the sample carrier 11 is large, depositing an equally large number (or substantially the same order) of barrier material pillars 31 on top of the sample carrier will naturally result in barrier material 31 being present between sets of first particles 21 and second particles 22. In the embodiment shown in Figure 4c, liquefying the barrier material 31 causes it to flow and fuse with the first particles 21 and second particles 22, so that interactions between the first particles 21 and second particles 22 can occur.

[0073] FIG. 5 illustrates an embodiment having a first layer 31 of barrier material containing first particles 21, a second layer 32 of barrier material containing second particles 22, and an intermediate layer 33 disposed between the first and second layers. The intermediate layer 33 can provide a buffer layer 33 that prevents direct contact between the first particles 21 and the second particles 22. This direct contact may be possible if the first layer is relatively thin, resulting in the first particles 21 being slightly misaligned and extending toward the outer surface of the layer. If the second layer 32 is applied directly on top of the first layer 31, the second particles 22 may already have interacted with the first particles 21 without any interaction being observed inside a charged particle microscope.

[0074] The first layer, second layer, and intermediate layer may all be similar. Alternatively, they may be selected to be different from one another. In one embodiment, the first material 31, the second material 32, and the intermediate layer 33 are all vitreous ice. Having the same material helps to locally liquefy the barrier materials 31, 32, and 33.

[0075] 6 shows an embodiment in which the first particles 21 are applied to the sample carrier 11 in the form of a barrier material layer 31 comprising the first particles 21. The second particles 22 are deposited on top of the barrier material 31 without being embedded in the respective barrier material.

[0076] FIG. 7 shows the embodiment of FIG. 6, but includes an intermediate layer 32 to prevent unintended interactions between the first particles 21 and the second particles 22.

[0077] Figure 8 shows an embodiment in which a number of first particles 21', 21" and second particles 22', 22" are deposited on top of a sample carrier, and barrier material is provided between the number of particles in the form of larger pillars 31. Similar to Figure 4c, it can be seen that barrier material 31 is provided between sets of first particles 21" and second particles 22". The barrier material 31 is provided such that the first particles 21" are spaced apart from the second particles 22", and the barrier material is provided between the first particles 21" and the second particles 22".

[0078] By liquefying the barrier material 31 of FIG. 8, the medium flows and entrains the first and second particles, which can result in interactions between the particles 21', 21'', 22', 22''.

[0079] 9 shows an embodiment similar to that of FIG. 8, but in this case the barrier material comprises heat absorbing elements 41 that help liquefy the barrier material 31. The heat absorbing elements 41 may comprise, for example, gold particles. By heating the heat absorbing elements 41 with a charged particle source, such as a laser or electron source, heat is acquired within the heat absorbing particles 41 and then released into the surrounding barrier material 31, which can then liquefy as a result of the heat imparted thereto. Once liquefied, the barrier material may comprise particles such that particle-particle interactions can occur.

[0080] Figure 10 shows an embodiment similar to Figure 3, but with a heat absorbing element 41, similar to Figure 9. This embodiment can be fabricated similarly to that described with respect to Figure 4, with the application of barrier material 31 including application of barrier material 31 including heat absorbing elements 41, such as gold nanoparticles.

[0081] 11 shows a further embodiment in which a sample 1 is prepared by providing a sample carrier 11 and then applying a layer of barrier material 31, for example, using standard cryo-EM sample preparation techniques known to those skilled in the art. After applying layer 31 and allowing the layer to solidify, first particles 21 and second particles 22 can be deposited on top of the barrier material layer, thereby providing barrier material 31 between first particles 21 and second particles 22. When barrier material 31 liquefies, first particles 21 and second particles 22 are captured by liquid barrier material 31 and can come into contact with each other to initiate an interaction.

[0082] 12 shows yet another embodiment of a sample 1, in which first particles 21 are deposited on a sample carrier 11 and a barrier material 31 containing second particles 22 is also deposited on the sample carrier 11. Here, the barrier material 31 is (at least partially) provided between the first particles 21 and the second particles 22, preventing the first particles 21 and the second particles 22 from coming into contact with each other.

[0083] Figure 13 shows an alternative to sample 1 of Figure 12, in which both the first particle 21 and the second particle 22 are embedded within a barrier material 31. A pillar of barrier material 31 containing each particle 21, 22 is then deposited on the sample carrier 11.

[0084] FIG. 15 shows an alternative to Sample 1 described by FIG. 11, but including an additional heat absorbing element 41.

[0085] In one embodiment, a method for time-resolved charged particle microscopy and a method for preparing a sample for the method use cryogenic temperatures to solidify a barrier material, the method comprising: 1) preparing a sample by forming an ice layer 31 on a cryogenic support 11 using cryo-deposition of (bio)molecules and / or particles 21, 22; 2) A period of thawing of the ice layer (barrier material 31) to allow for dynamics in the liquid phase, especially interactions of molecules / particles 21, 22, followed by 3) refreezing (revitrifying) the barrier material 31 to freeze the final state in vitreous ice for cryo-EM imaging.

[0086] To deposit particles onto the sample 1, soft landing deposition [1] or shock freeze deposition [2] can be used to deposit hydrated or ice-covered biomolecules and / or particles onto a bare support 11 (see Figures 4a, 8, 9, 12, 13) or onto a support 11 pre-covered with ice 31 or a sample in vitreous ice (see Figures 6, 7, 11, 15).

[0087] These deposition methods allow for the sequential or simultaneous deposition of multiple reactants (particles 21, 22) in a cold or ice-embedded state without substantial kinetic, mixing, and / or chemical reactions.

[0088] By using a subsequent ultrashort heating step to bring the deposited frozen sample into a liquid phase, the reactant molecules or particles can diffuse and react.

[0089] Since the bulk of the carrier remains cold, the liquefied barrier material quickly freezes (re-vitrifies) after heating is stopped. Brief thawing and re-vitrification, for example by application of short laser heating, is known to those skilled in the art [3-6]. Other heating methods can be used as well. For example, the sample carrier 11 may be equipped with an integrated microheater (not shown) that can (locally) heat the grid 11 to liquefy the barrier material 31.

[0090] The method according to the invention enables time-resolved biochemical experiments using charged particle microscopy, in particular cryo-EM. The cryo-deposition of the sample prevents dynamics and reactions, while the subsequent melting and re-vitrification steps allow dynamics and reactions, providing a defined t=0s state and temporal control.

[0091] References [1]A Preparative Mass Spectrometer to Deposit Intact Large Native Protein Complexes,Paul Fremdling,Tim K.Esser,Bodhisattwa Saha,Alexander A.Makarov,Kyle L.Fort,Maria Reinhardt-Szyba,Joseph Gault,and Stephan Rauschenbach,,ACS Nano2022,16,1444314455 [2]Controlled beams of shock-frozen,isolated,biological and artificial nanoparticles,Amit K.Samanta,Muhamed Amin,Armando D.Estillore,Nils Roth,Lena Worbs,Daniel A.Horke,and Jochen Kupper,Structural Dynamics 7,024304(2020)

Claims

1. 1. A method of time-resolved charged particle microscopy comprising: - providing a sample for charged particle microscopy, said sample comprising first particles and second particles, said sample comprising a barrier material between said first particles and said second particles; - liquefying at least a portion of the barrier material to allow interaction between the first particles and the second particles; and - observing the resulting interaction between the first particle and the second particle in a charged particle microscope.

2. The method of claim 1 , wherein liquefying the barrier material comprises heating the barrier material.

3. The method of claim 2 wherein a laser is used to liquefy the barrier material.

4. The method of claim 2 , wherein a charged particle beam is used to liquefy the barrier material.

5. 5. The method of claim 4, wherein the sample comprises heat absorbing particles, in particular gold particles, and the charged particle beam is used to heat the heat absorbing particles for subsequent liquefaction of the barrier material.

6. The method of any one of claims 1 to 5, further comprising the step of re-solidifying the barrier material after the step of liquefying the barrier material.

7. The method of claim 6 , wherein re-solidifying the barrier material comprises cooling the barrier material.

8. 8. The method of claim 6 or 7, comprising, after the re-solidifying step, a subsequent step of re-liquefying at least a portion of the barrier material, and thereafter observing the resulting interaction between the first particles and the second particles.

9. 9. The method according to claim 1, wherein at least one of the first particle and the second particle is selected from the group consisting of a nanoparticle, a biomolecule, a virus, a protein, a pharmaceutical molecule, and a ligand.

10. 10. The method of any one of claims 1 to 9, wherein the sample is a cryo-electron microscope sample and the barrier material comprises amorphous ice.

11. The method of claim 10 , wherein liquefying the barrier material is performed when the sample for charged particle microscopy is placed inside the charged particle microscope.

12. 1. A method of preparing a sample for time-resolved charged particle microscopy, comprising: - providing a sample carrier for charged particle microscopy; - depositing first particles, second particles and a barrier material on the sample support, the order in which the first particles, second particles and barrier material are deposited is such that the first particles are provided at a distance from the second particles and the barrier material is provided between the first particles and the second particles.

13. The method of claim 12 , wherein the barrier material is deposited on the sample support prior to the step of depositing the second particles.

14. 14. The method of claim 12 or 13, wherein the barrier material is deposited on the sample support before the step of depositing the second material.

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